University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Journal of Biological Chemistry

Citations 61

"Development And Fertility In Caenorhabditis Elegans Clk-1 Mutants Depend Upon Transport Of Dietary Coenzyme Q8 To Mitochondria"
J. Biol. Chem. 2002 Volume 277, Issue 47 Pages 45020-45027
Tanya Jonassen, Beth N. Marbois, Kym F. Faull, Catherine F. Clarke, and Pamela L. Larsen

Abstract: The Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and instead accumulate the biosynthetic intermediate demethoxy-Q9 (DMQ9). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q8-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q8-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ9 are high in the clk-1(qm30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q8 present is assimilated from the E. coli. Furthermore, this E. coli-specific Q8 is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q8 to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.

"Kappa -Hefutoxin1, A Novel Toxin From The Scorpion Heterometrus Fulvipes With Unique Structure And Function. IMPORTANCE OF THE FUNCTIONAL DIAD IN POTASSIUM CHANNEL SELECTIVITY"
J. Biol. Chem. 2002 Volume 277, Issue 33 Pages 30040-30047
Kellathur N. Srinivasan, Vaithiyalingam Sivaraja, Isabelle Huys, Toru Sasaki, Betty Cheng, Thallampuranam Krishnaswamy S. Kumar, Kazuki Sato, Jan Tytgat, Chin Yu, B. Chia Cheng San, Shoba Ranganathan, H. John Bowie, R. Manjunatha Kini, and Ponnampalam Gopalakrishnakone

Abstract: An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, [kappa]-hefutoxin1 and [kappa]-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that [kappa]-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β[-]sheets. Based on the presence of the functional diad (Tyr5/Lys19) at a distance (6.0 ± 1.0 A) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. [kappa]-Hefutoxin 1 not only blocks the voltage-gated K+-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of [kappa]-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

"Evidence Against Glycogen Cycling Of Gluconeogenic Substrates In Various Liver Preparations"
J. Biol. Chem. 2002 Volume 277, Issue 32 Pages 28648-28655
Keld Fosgerau, Jens Breinholt, James G. McCormack, and Niels Westergaard

Abstract: The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-D-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-D-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-D-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 ± 0.19 versus 0.33 ± 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 ± 0.09 versus 0.18 ± 0.08, p = not significant) (1,4-dideoxy-1,4-imino-D-arabinitol versus vehicle, µmol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-D-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (µmol of glycosyl units/g of wet liver) were similar in fed (235 ± 19 versus 217 ± 22, p = not significant) or fasted rats (10 ± 2 versus 7 ± 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-D-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-D-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes.

"Heme Nitrosylation Of Deoxyhemoglobin By S-Nitrosoglutathione Requires Copper"
J. Biol. Chem. 2002 Volume 277, Issue 27 Pages 24135-24141
Andrea A. Romeo, John A. Capobianco, and Ann M. English

Abstract: Nitrous oxide (NO) reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1,10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N,N'',N''-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of ~100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N'',N''-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysβ93 of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.'

"Candoxin, A Novel Toxin From Bungarus Candidus, Is A Reversible Antagonist Of Muscle (α β γ δ) But A Poorly Reversible Antagonist Of Neuronal Alpha 7 Nicotinic Acetylcholine Receptors"
J. Biol. Chem. 2002 Volume 277, Issue 20 Pages 17811-17820
Selvanayagam Nirthanan, Eric Charpantier, Ponnampalam Gopalakrishnakone, Matthew C. E. Gwee, Hoon-Eng Khoo, Li-Sam Cheah, Daniel Bertrand, and R. Manjunatha Kini

Abstract: In contrast to most short and long chain curaremimetic neurotoxins that produce virtually irreversible neuromuscular blockade in isolated nerve-muscle preparations, candoxin, a novel three-finger toxin from the Malayan krait Bungarus candidus, produced postjunctional neuromuscular blockade that was readily and completely reversible. Nanomolar concentrations of candoxin (IC50 = ~10 nM) also blocked acetylcholine-evoked currents in oocyte-expressed rat muscle (αβγδ) nicotinic acetylcholine receptors in a reversible manner. In contrast, it produced a poorly reversible block (IC50 = ~50 nM) of rat neuronal α7 receptors, clearly showing diverse functional profiles for the two nicotinic receptor subsets. Interestingly, candoxin lacks the helix-like segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long chain neurotoxins, reported to be critical for binding to α7 receptors. However, its solution NMR structure showed the presence of some functionally invariant residues involved in the interaction of both short and long chain neurotoxins to muscle (αβγδ) and long chain neurotoxins to α7 receptors. Candoxin is therefore a novel toxin that shares a common scaffold with long chain α-neurotoxins but possibly utilizes additional functional determinants that assist in recognizing neuronal α7 receptors.

"Plasmodium Falciparum Histidine-rich Protein-2 (PfHRP2) Modulates The Redox Activity Of Ferri-protoporphyrin IX (FePPIX). PEROXIDASE-LIKE ACTIVITY OF THE PfHRP2-FePPIX COMPLEX"
J. Biol. Chem. 2002 Volume 277, Issue 17 Pages 14514-14520
Ryuichi Mashima, Leann Tilley, Mary-Anne Siomos, Vicki Papalexis, Mark J. Raftery, and Roland Stocker

Abstract: Histidine-rich protein-2 from Plasmodium falciparum (PfHRP2) binds up to 50 molecules of ferri-protoporphyrin IX (FePPIX) (Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T., and Marletta, M. A. (1999) Biochemistry 38, 16916-16924). We reasoned that the PfHRP2-FePPIX complex has antioxidant properties that could be beneficial to the parasite. Therefore, we examined whether binding to PfHRP2 modulated the redox properties of FePPIX. We observed that PfHRP2 completely inhibited the auto-oxidation of ascorbate mediated by free FePPIX. We also investigated the peroxidase activity of PfHRP2-FePPIX using 13-hydroperoxy-9,11-octadienoate (18:2-OOH) as substrate. Reaction of PfHRP2-FePPIX with 18:2-OOH in the presence of added reducing agents gave 13-hydroxy-9,11-octadienoate (18:2-OH) as a major product and 13-keto-9,11-octadienoate (18:2=O) and 9,12,13-trihydroxy-10-octadecaenoate as minor products. Binding of FePPIX to PfHRP2 lowered the rate of decomposition of 18:2-OOH and increased the 18:2-OH to 18:2=O ratio. Similar to other authentic peroxidases, phenols, amines, and biological reductants like ascorbate promoted 18:2-OH production, and NaCN inhibited 18:2-OH production. Thioanisole also acted as a reductant and was converted to thioanisole sulfoxide, suggesting formation of compound I during the reaction. These data show that PfHRP2 modulates the redox activity of FePPIX and that the PfHRP2-FePPIX complex may have previously unrecognized antioxidant properties.

"Epoxyisoprostane And Epoxycyclopentenone Phospholipids Regulate Monocyte Chemotactic Protein-1 And Interleukin-8 Synthesis. Formation Of These Oxidized Phospholipids In Response To Interleukin-1β"
J. Biol. Chem. 2002 Volume 277, Issue 9 Pages 7271-7281
Ganesamoorthy Subbanagounder, Jason W. Wong, Hans Lee, Kym F. Faull, Elizabeth Miller, Joseph L. Witztum, and Judith A. Berliner

Abstract: Monocyte recruitment to the vessel wall, mediated by monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8), plays an important role in atherogenesis. We have shown previously that minimally oxidized low density lipoprotein, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), activates endothelial cells to produce MCP-1 and IL-8. By using liquid chromatography/mass spectrometry methods coupled with bioassay, we report a family of epoxyisoprostane (PEIPC) and epoxycyclopentenone (PECPC) phospholipids that are the components of Ox-PAPC responsible for the majority of this activity. Ox-PAPC contains five chromatographically distinguishable active PEIPC components (m/z 825.5) and four PECPC components (m/z 810.5). All nine components induced endothelial cell synthesis of IL-8 and MCP-1 in a dose-dependent fashion between 0.1 and 5 µM concentrations. The five PEIPC components had identical functional groups and all underwent dehydration to produce m/z 810.5. We present evidence that these phospholipids are regioisomers with epoxide groups at the 5,6-, 8,9-, 11,12-, or 14,15-positions of the sn-2 fatty acid and their epoxide groups is important for biological activity. We have shown previously that peroxisome proliferator-activated receptor α is involved in MCP-1 synthesis in response to Ox-PAPC. We now show that PEIPC and PECPC isomers are potent activators of peroxisome proliferator-activated receptor α. PEIPC and PECPC isomers are strongly recognized by specific circulating murine natural autoantibodies (EO6) and accumulate in cells treated with IL-1β. These studies demonstrate that PEIPC and PECPC isomers are potent activators of endothelial cells increasing synthesis of IL-8 and MCP-1. Their accumulation in cells exposed to cytokines and in atherosclerotic lesions suggests that these lipids may play a role in a number of chronic disease processes.

"Identification Of A New Class Of Molecules, The Arachidonyl Amino Acids, And Characterization Of One Member That Inhibits Pain"
J. Biol. Chem. 2001 Volume 276, Issue 46 Pages 42639-42644
Susan M. Huang, Tiziana Bisogno, Timothy J. Petros, Sai Y. Chang, Paul A. Zavitsanos, Robert E. Zipkin, Ramachandran Sivakumar, Andrew Coop, Dean Y. Maeda, Luciano De Petrocellis, Sumner Burstein, Vincenzo Di Marzo, and J. Michael Walker

Abstract: In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl γ-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals.

"Structure Of A Neutral Glycosphingolipid Recognized By Human Antibodies In Polyagglutinable Erythrocytes From The Rare NOR Phenotype"
J. Biol. Chem. 2001 Volume 276, Issue 44 Pages 40574-40582
Maria Duk, Bruce B. Reinhold, Vernon N. Reinhold, Grazyna Kusnierz-Alejska, and Elwira Lisowska

Abstract: NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an α-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galα1[right-arrow]3Gal- sequence and that anti-NOR agglutinins were common human anti-Galα1[right-arrow]3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with α-galactosidase and β-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an α-galactosylated Gb4Cer with a β-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1[right-arrow]4HexN1[right-arrow]3Hex1[right-arrow]4Hex1[right-arrow]4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galα1[right-arrow]4GalNAcβ1[right-arrow]3Galα1[right-arrow]4Galβ1[right-arrow]4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galα1[right-arrow]3Gal xenoantibodies.

"Polyunsaturated Eicosapentaenoic Acid Displaces Proteins From Membrane Rafts By Altering Raft Lipid Composition"
J. Biol. Chem. 2001 Volume 276, Issue 40 Pages 37335-37340
Thomas M. Stulnig, Joakim Huber, Norbert Leitinger, Esther-Maria Imre, Pavla Angelisov&aacute;, Peter Nowotny, and Werner Waldh&auml;usl

Abstract: Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.

"Hormone-mediated Dephosphorylation Of Specific Histone H1 Isoforms"
J. Biol. Chem. 2001 Volume 276, Issue 39 Pages 36467-36473
Geoffrey C. Banks, Leesa J. Deterding, Kenneth B. Tomer, and Trevor K. Archer

Abstract: We have previously shown a connection between histone H1 phosphorylation and the transcriptional competence of the hormone inducible mouse mammary tumor virus (MMTV) promoter. Prolonged exposure of mouse cells to dexamethasone concurrently dephosphorylated histone H1 and rendered the MMTV promoter refractory to hormonal stimulation and, therefore, transcriptionally unresponsive. Using electrospray mass spectrometry, we demonstrate here that prolonged dexamethasone treatment differentially effects a subset of the six somatic H1 isoforms in mouse cells. H1 isoforms H1.0, H1.1, and H1.2 are non-responsive to hormone whereas prolonged dexamethasone treatment effectively dephosphorylated the H1.3, H1.4, and H1.5 isoforms. The protein kinase inhibitor staurosporine, shown to dephosphorylate histone H1 and down-regulate MMTV in cultured cells, appears only to completely dephosphorylate the H1.3 isoform. These results suggest that dephosphorylation of specific histone H1 isoforms may contribute to the previously observed decrease in transcriptional competence of the MMTV promoter through the modulation of chromatin structure. In a broader sense, this work advances the hypothesis that post-translational modifications of individual histone H1 isoforms directly influence the transcriptional activation/repression of specific genes.

"Mass Spectral Analysis Of Protein-based Radicals Using DBNBS. NONRADICAL ADDUCT FORMATION VERSUS SPIN TRAPPING"
J. Biol. Chem. 2001 Volume 276, Issue 24 Pages 21022-21027
Angelo Filosa and Ann M. English

Abstract: Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H2O2 were investigated by electrospray mass spectrometry (ESI-MS) using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS). Up to four DBNBS-cyt c adducts were observed in the mass spectra. However, by varying the reaction conditions (0-5 molar equivalents of H2O2 and substituting cyt c with its cyanide adduct which is resistant to peroxidation), noncovalent DBNBS adduct formation was inferred. Nonetheless, optical difference spectra revealed the presence of a small fraction of covalently trapped DBNBS. To probe the nature of the noncovalent DBNBS adducts, the less basic proteins, metmyoglobin (Mb) and α-lactalbumin, were substituted for cyt c in the cyt c/H2O2/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H2O2/DBNBS reactions, whereas no adducts were detected following α-lactalbumin/H2O2/DBNBS incubation, which is consistent with adduct formation via spin trapping only. Titration with DBNBS at pH 2.0 yielded noncovalent DBNBS-cyt c adducts and induced folding of acid-denatured cyt c, as monitored by ESI-MS and optical spectroscopy, respectively. Thus, the noncovalent DBNBS-cyt c mass adducts observed are assigned to ion pair formation occurring between the negatively charged sulfonate group on DBNBS and positively charged surface residues on cyt c. The results reveal the pitfalls inherent in using mass spectral data with negatively charged spin traps such as DBNBS to identify sites of radical formation on basic proteins such as cyt c.

"Oxidized Alkyl Phospholipids Are Specific, High Affinity Peroxisome Proliferator-activated Receptor γ Ligands And Agonists"
J. Biol. Chem. 2001 Volume 276, Issue 19 Pages 16015-16023
Sean S. Davies, Aaron V. Pontsler, Gopal K. Marathe, Kathleen A. Harrison, Robert C. Murphy, Jerald C. Hinshaw, Glenn D. Prestwich, Andy St. Hilaire, Stephen M. Prescott, Guy A. Zimmerman, and Thomas M. McIntyre

Abstract: Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A1 digestion, that PPARγ agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARγ. [3H]azPC bound recombinant PPARγ with an affinity (Kd(app) ~40 nM) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nM. Overexpression of PPARα or PPARγ revealed that azPC was a specific PPARγ agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARγ that induce PPAR-responsive genes.

"Novel Intracellular Sb-V Reducing Activity Correlates With Antimony Susceptibility In Leishmania Donovani"
J. Biol. Chem. 2001 Volume 276, Issue 6 Pages 3971-3976
Pninit Shaked-Mishan, Nina Ulrich, Moshe Ephros, and Dan Zilberstein

Abstract: The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb-V). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-RIS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. Amastigotes (the intracellular form) and promastigotes (the extracellular form) accumulate Sb-V and Sb-III via separate transport systems. Stage-specific intracellular Sb-V reducing activity was apparent in amastigotes, which reduced the negligibly toxic Sb-V to highly toxic Sb-III. This amastigote-specific reducing activity was deficient in the Pentostam-resistant mutant L. donovani Ld1S.20. These data indicate that parasite susceptibility to Sb-V correlates with its level of Sb-V reducing activity. Also, in promastigotes of both wild-type L. donovani and the Pentostam-resistant mutant L. donovani Ld1S.20, Sb-V inhibited the toxicity of Sb-III but not of Asm. Both Sb-V and Sb-III were toxic to wild-type amastigotes. However, as observed in promastigotes, in mutant amastigotes Sb-V inhibits Sb-III but not As-III activity, Anion exchange chromatography showed that intracellular antimony metabolism occurred in both promastigotes and amastigotes. These data demonstrate that the interaction between the two antimony oxidation states occurs intracellularly, within the parasite. The results also indicate that Sb-V anti-leishmanial activity is dependent on its reduction to Sb-III. The mechanism of this novel intracellular Sb-V reduction has yet to be identified, and it may or may not be enzymatic, This is the first description of intracellular Sb-V reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.

"Human Apolipoprotein C-I Accounts For The Ability Of Plasma High Density Lipoproteins To Inhibit The Cholesteryl Ester Transfer Protein Activity"
J. Biol. Chem. 2000 Volume 275, Issue 48 Pages 37504-37509
Thomas Gautier, David Masson, Jean-Paul Pais de Barros, Anne Athias, Philippe Gambert, Dominique Aunis, Marie-H&eacute;l&eagrave;ne Metz-Boutigue, and Laurent Lagrost

Abstract: The aim of the present study was to identify the protein that accounts for the cholesteryl ester transfer protein (CETP)-inhibitory activity that is specifically associated with human plasma high density lipoproteins (HDL). To this end, human HDL apolipoproteins were fractionated by preparative polyacrylamide gradient gel electrophoresis, and 30 distinct protein fractions with molecular masses ranging from 80 down to 2 kDa were tested for their ability to inhibit CETP activity. One single apolipoprotein fraction was able to completely inhibit CETP activity. The N-terminal sequence of the 6-kDa protein inhibitor matched the N-terminal sequence of human apoC-I, the inhibition was completely blocked by specific anti-apolipoprotein C-I antibodies, and mass spectrometry analysis confirmed the identity of the isolated inhibitor with full-length human apoC-I. Pure apoC-I was able to abolish CETP activity in a concentration-dependent manner and with a high efficiency (IC50 = 100 nmol/liter). The inhibitory potency of total delipidated HDL apolipoproteins completely disappeared after a treatment with anti-apolipoprotein C-I antibodies, and the apoC-I deprivation of native plasma HDL by immunoaffinity chromatography produced a mean 43% rise in cholesteryl ester transfer rates. The main localization of apoC-I in HDL and not in low density lipoprotein in normolipidemic plasma provides further support for the specific property of HDL in inhibiting CETP activity.

"Isolation And Characterization Of Gomesin, An 18-Residue Cysteine-rich Defense Peptide From The Spider Acanthoscurria Gomesiana Hemocytes With Sequence Similarities To Horseshoe Crab Antimicrobial Peptides Of The Tachyplesin Family"
J. Biol. Chem. 2000 Volume 275, Issue 43 Pages 33464-33470
Pedro I. Silva, Jr., Sirlei Daffre, and Philippe Bulet

Abstract: We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine α-amide, and four cysteine residues forming two disulfide bridges. This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions. Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes. Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.

"Molecular Characterization Of Lantibiotic-synthesizing Enzyme EpiD Reveals A Function For Bacterial Dfp Proteins In Coenzyme A Biosynthesis"
J. Biol. Chem. 2000 Volume 275, Issue 41 Pages 31838-31846
Thomas Kupke, Michael Uebele, Dietmar Schmid, G&uuml;nther Jung, Michael Blaesse, and Stefan Steinbacher

Abstract: The lantibiotic-synthesizing flavoprotein EpiD catalyzes the oxidative decarboxylation of peptidylcysteines to peptidyl-aminoenethiols. The sequence motif responsible for flavin coenzyme binding and enzyme activity is conserved in different proteins from all kingdoms of life. Dfp proteins of eubacteria and archaebacteria and salt tolerance proteins of yeasts and plants belong to this new family of flavoproteins. The enzymatic function of all these proteins was not known, but our experiments suggested that they catalyze a similar reaction like EpiD and/or may have similar substrates and are homododecameric flavoproteins. We demonstrate that the N-terminal domain of the Escherichia coli Dfp protein catalyzes the decarboxylation of (R)-4-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This reaction is essential for coenzyme A biosynthesis.'

"The Biosynthesis Of A2E, A Fluorophore Of Aging Retina, Involves The Formation Of The Precursor, A2-PE, In The Photoreceptor Outer Segment Membrane"
J. Biol. Chem. 2000 Volume 275, Issue 38 Pages 29354-29360
Jianghua Liu, Yasuhiro Itagaki, Shimon Ben-Shabat, Koji Nakanishi, and Janet R. Sparrow

Abstract: The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-L-α-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C77H124O8PN) accompanied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.

"Crocus Sativus Lectin Recognizes Man(3)GlcNAc In The N-glycan Core Structure"
J. Biol. Chem. 2000 Volume 275, Issue 35 Pages 26772-26779
Tapas K. Hazra, Tadahide Izumi, Radhika Venkataraman, Yoke W. Kow, Miral Dizdaroglu, and Sankar Mitra

Abstract: Crocus sativus lectin (CSL) is one of the truly mannose-specific plant lectins that has a unique binding specificity that sets it apart from others. We studied sugar-binding specificity of CSL in detail by a solution phase method (fluorescence polarization) and three solid phase methods (flow injection, surface plasmon resonance, and microtiter plate), using a number of different glycopeptides and oligosaccharides. CSL binds the branched mannotriose structure in the N-glycan core. Substitution of the terminal Man in the Man α(1-3)Man branch with GlcNAc drastically decreases binding affinity much more than masking of the terminal Man in the Man α(l-G)Man branch, Most interestingly, the β-Man-linked GlcNAc in N-glycan core structure contributes greatly to the binding. The effect of this GlcNAc is so strong that it can substantially offset the negative effect of substitution on the nonreducing terminal Man residues. On the other hand, the GlcNAc that is usually attached to Asn in N-glycans and the L-FUC linked at the 6-position of the GlcNAc are irrelevant to the binding. A bisecting GlcNAc neither contributes to nor interferes with the binding. This unique binding specificity of CSL offers many possibilities of its use in analytical and preparative applications.

"Kinetic And Structural Characterization Of The Glutathione-binding Site Of Aldose Reductase"
J. Biol. Chem. 2000 Volume 275, Issue 28 Pages 21587-21595
Bharat L. Dixit, Ganesaratnam K. Balendiran, Stanley J. Watowich, Sanjay Srivastava, Kota V. Ramana, J. Mark Petrash, Aruni Bhatnagar, and Satish K. Srivastava

Abstract: Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000 fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.

"Mechanism Of Reaction Of Myeloperoxidase With Nitrite"
J. Biol. Chem. 2000 Volume 275, Issue 27 Pages 20597-20601
Ursula Burner, Paul G. Furtm&uuml;ller, Anthony J. Kettle, Willem H. Koppenol, and Christian Obinger

Abstract: Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre- steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15°C the rate of formation of the low spin MPO-nitrite complex is (2.5 ± 0.2) x 104 M-1 s-1 at pH 7 and (2.2 ± 0.7) x 106 M-1 s-1 at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 ± 0.1 mM at pH 7 and 31.3 ± 0.5 ?M at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second- order rate constant of reduction of compound I to compound II at 15°C is (2.0 ± 0.2) x 106 M-1 s-1 at pH 7 and (1.1 ± 0.2) x 107 M-1 s-1 at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15°C is (5.5 ± 0.1) x 102 M-1 s-1 at pH 7 and (8.9 ± 1.6) x 104 M-1 S-1 at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 ± 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.

"Thrombocidins, Microbicidal Proteins From Human Blood Platelets, Are C-terminal Deletion Products Of CXC Chemokines"
J. Biol. Chem. 2000 Volume 275, Issue 27 Pages 20374-20381
Jeroen Krijgsveld, Sebastian A. J. Zaat, Jan Meeldijk, Peter A. van Veelen, Gang Fang, Bert Poolman, Ernst Brandt, Jan E. Ehlert, Alma J. Kuijpers, Gerard H. M. Engbers, Jan Feijen, and Jacob Dankert

Abstract: Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their α-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

"Nitric Oxide Modulates A Late Step Of Exocytosis"
J. Biol. Chem. 2000 Volume 275, Issue 27 Pages 20274-20279
Jos&eacute; D. Machado, Fernando Segura, Miguel A. Brioso, and Ricardo Borges

Abstract: The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba2+-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N[omega]-nitro-L-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba2+. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

"Xenobiotic-metabolizing Cytochromes P450 Convert Prostaglandin Endoperoxide To Hydroxyheptadecatrienoic Acid And The Mutagen, Malondialdehyde"
J. Biol. Chem. 2000 Volume 275, Issue 16 Pages 11784-11790
John P. Plastaras, F. Peter Guengerich, Daniel W. Nebert, and Lawrence J. Marnett

Abstract: Cyclooxygenases catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides. Cyclooxygenase-2- and the xenobiotic-metabolizing cytochrome P450s 1A and 3A are all aberrantly expressed during colorectal carcinogenesis. To probe for a role of P450s in prostaglandin endoperoxide metabolism, we studied the 12-hydroxyheptadecatrienoate (HHT)/malondialdehyde (MDA) synthase activity of human liver microsomes and purified P450s. We found that human liver microsomes have HHT/MDA synthase activity that is concentration-dependent and inhibited by the P450 inhibitors, ketoconazole and clotrimazole with IC50 values of 1 and 0.4 µM, respectively. This activity does not require P450 reductase. HHT/MDA synthase activity was present in purified P450s but not in heme alone or other heme proteins. The catalytic activities of various purified P450s were determined by measuring rates of MDA production from prostaglandin endoperoxide. At 50 µM substrate, the catalytic activities of purified human P450s varied from 10±1 to 0.62±0.02 min-1, 3A4>>2E1>1A2. Oxabicycloheptane analogs of prostaglandin endoperoxide, U-44069 and U-46619, induced spectral changes in human P450 3A4 with Ks values of 240±20 and 130±10 µM, respectively. These results suggest that co-expression of cyclooxygenase-2 and P450s in developing cancers may contribute to genomic instability due to production of the endogenous mutagen, MDA.

"Mapping Of The 5-2-Deoxyribose-5-phosphate Lyase Active Site In DNA Polymerase β By Mass Spectrometry'"
J. Biol. Chem. 2000 Volume 275, Issue 14 Pages 10463-10471
Leesa J. Deterding, Rajendra Prasad, Gregory P. Mullen, Samuel H. Wilson, and Kenneth B. Tomer

Abstract: The mechanism of the 5-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA β-polymerase (β-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyzes. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyzes. The 8-kDa domain of β-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys72 residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys72 as the sole Schiff base nucleophile in the 8-kDa domain of β-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys72 at the abasic site.'

"Vesicular Ca2+ Participates In The Catalysis Of Exocytosis"
J. Biol. Chem. 2000 Volume 275, Issue 13 Pages 9136-9142
Michelle L. Mundorf, Kevin P. Troyer, Spencer E. Hochstetler, Joseph A. Near, and R. Mark Wightman

Abstract: Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracellular Ca2+ evoked by depolarizing agents. Subcellular studies revealed that reserpine and tetrabenazine at concentrations near their Ki values not only could increase cytoplasmic catecholamines but also could displace Ca2+ from vesicles. Furthermore, transient exposure to tetrabenazine and reserpine, but not methyl reserpate and reserpic acid, induced exocytotic release of catecholamines. Reserpine induced a rise in intracellular Ca2+, as detected by whole-cell measurements with Fura-2. It could induce exocytosis, albeit at a lower frequency, in Ca2+-free solutions, supporting an internal Ca2+ source. Depletion of endoplasmic reticulum and mitochondrial Ca2+ pools did not eliminate the reserpine-activated release. These results indicate that vesicular Ca2+ can play an important role in exocytosis and under some conditions may be involved in initiating this process.

"Cloning And Characterization Of A New Member Of The Nudix Hydrolases From Human And Mouse"
J. Biol. Chem. 2000 Volume 275, Issue 12 Pages 8844-8853
Hanjing Yang, Malgorzata M. Slupska, Ying-Fei Wei, Jennifer H. Tai, Wendy M. Luther, Yu-Rong Xia, Diana M. Shih, Ju-Huei Chiang, Claudia Baikalov, Sorel Fitz-Gibbon, Isabella T. Phan, Alexis Conrad, and Jeffrey H. Miller

Abstract: Proteins containing the Nudix box ''GX5EX7REUXEEXGU'' (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with Km values of 32 and 83 µM, respectively; the Vmax for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

"Correlated Oscillations In Glucose Consumption, Oxygen Consumption, And Intracellular Free Ca2+ In Single Islets Of Langerhans"
J. Biol. Chem. 2000 Volume 275, Issue 9 Pages 6642-6650
Sung-Kwon Jung, Lisa M. Kauri, Wei-Jun Qian, and Robert T. Kennedy

Abstract: Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 ± 0.2 min and amplitude of 0.8 ± 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca2+ and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca2+ ([Ca2+]i) revealed that decreases in [Ca2+]i preceded increases in glucose consumption by 7.4 ± 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca2+]i preceded increases in oxygen consumption by 1.5 ± 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca2+ has stopped entering the cell. Glycolysis stimulates further Ca2+ entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca2+]i. A model to explain these results is described.

"Structural Characterization Of The Inflammatory Moiety Of A Variable Major Lipoprotein Of Borrelia Recurrentis"
J. Biol. Chem. 2000 Volume 275, Issue 2 Pages 937-941
Ian G. Scragg, Dominic Kwiatkowski, Vincent Vidal, Andrew Reason, Thanai Paxton, Maria Panico, Ann Dell, and Howard Morris

Abstract: Louse-borne relapsing fever, caused by Borrelia recurrentis, provides one of the best documented examples of the causative role of tumor necrosis factor (TNF) in the pathology of severe infection in humans. We have identified the principal TNF-inducing factor of B. recurrentis as a variable major lipoprotein (Vmp). Here we report the complete gene sequence of Vmp, including its lipoprotein leader sequence. Using metabolically labeled forms of the native Vmp we confirm that the TNF inducing properties are associated with the lipid portion of the molecule. Quadrupole orthogonal time of flight mass spectrometry unequivocally locates the lipidic moiety at the NH2-terminal cysteine of the native polypeptide, and indicates the existence of three forms which are consistent with the structures C16:0, C16:0, C16:0 glyceryl cysteine; C18:1, C16:0, C16:0 glyceryl cysteine; and C18:0, C16:0, C16:0 glyceryl cysteine. These data provide the first direct evidence that the TNF inducing lipid modification of native Borrelia lipoproteins is a structural homologue of the murein lipoprotein of Escherichia coli.

"Structural Identification Of A Novel Pro-inflammatory Epoxyisoprostane Phospholipid In Mildly Oxidized Low Density Lipoprotein"
J. Biol. Chem. 1999 Volume 274, Issue 35 Pages 24787-24798
Andrew D. Watson, Ganesamoorthy Subbanagounder, Derek S. Welsbie, Kym F. Faull, Mohamad Navab, Michael E. Jung, Alan M. Fogelman, and Judith A. Berliner

Abstract: One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A2 and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.

"Multiple Enzymatic Activities Of The Murein Hydrolase From Staphylococcal Phage Phi 11. IDENTIFICATION OF A D-ALANYL-GLYCINE ENDOPEPTIDASE ACTIVITY"
J. Biol. Chem. 1999 Volume 274, Issue 22 Pages 15847-15856
William Wiley Navarre, Hung Ton-That, Kym F. Faull, and Olaf Schneewind

Abstract: Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages φ11, 80α, 187, Twort, and φPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the φ11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the φ11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, φ11(δ181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.

"Equilibrium Binding Studies Of Non-claret Disjunctional Protein (Ncd) Reveal Cooperative Interactions Between The Motor Domains"
J. Biol. Chem. 1998 Volume 273, Issue 52 Pages 35307-35318
Kelly A. Foster, John J. Correia, and Susan P. Gilbert

Abstract: Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-β, γ-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
Binding affinity Bacteria Stopped-flow Kinetic

"Electron Paramagnetic Resonance Detection Of Free Tyrosyl Radical Generated By Myeloperoxidase, Lactoperoxidase, And Horseradish Peroxidase"
J. Biol. Chem. 1998 Volume 273, Issue 48 Pages 32030-32037
Michael L. McCormick, Joseph P. Gaut, Tien-Sung Lin, Bradley E. Britigan, Garry R. Buettner, and Jay W. Heinecke

Abstract: Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on L-tyrosine in vitro, it forms o,o-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free tyrosyl radical (a2,6H = 6.3 G, a3,5H = 1.6 G, and aβH = 15.0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN = 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN = 15.6 G, a13C () = 8.0 G, a13C () = 7.1 G, a13C () = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring of L-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and L-tyrosine residues to the myeloperoxidase-H2O2-L-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2 to convert L-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of L-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.'

"Anchor Structure Of Staphylococcal Surface Proteins. II. COOH-TERMINAL STRUCTURE OF MURAMIDASE AND AMIDASE-SOLUBILIZED SURFACE PROTEIN"
J. Biol. Chem. 1998 Volume 273, Issue 44 Pages 29135-29142
William Wiley Navarre, Hung Ton-That, Kym F. Faull, and Olaf Schneewind

Abstract: Surface proteins of the Gram-positive organism Staphylococcus aureus are anchored to the bacterial cell wall by a transpeptidation mechanism during which the polypeptide is cleaved between the threonine (T) and the glycine (G) of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino of the pentaglycyl cross-bridge within the staphylococcal peptidoglycan. Previous work examined the anchor structure of surface proteins solubilized from the peptidoglycan by treatment with lysostaphin or φ11 hydrolase and identified COOH-terminally linked triglycyl or L-Ala-D-iGln-L-Lys(Gly5)-D-Ala and MurNAc-[L-Ala-D-iGln-L-Lys(Gly5)-D-Ala](β1-4)-GlcNAc, respectively. Here, we report the anchor structure of surface proteins solubilized with N-acetylmuramidase and N-acetylmuramyl-L-alanine amidase. N-Acetylmuramidase-released surface protein was linked to MurNAc-[L-Ala-D-iGln-L-Lys(Gly5)-D-Ala](β1-4)-GlcNAc, whereas N-acetylmuramyl-L-alanine amidase treatment of the cell wall solubilized surface proteins linked to L-Ala-D-iGln-L-Lys(Gly5)-D-Ala. Most, but not all, anchor structures were cross-linked to other cell wall subunits, in which the D-alanyl at position four was amide linked to the pentaglycyl of a neighboring wall peptide.

"The Dependence Of Membrane Permeability By The Antibacterial Peptide Cecropin B And Its Analogs, CB-1 And CB-3, On Liposomes Of Different Composition"
J. Biol. Chem. 1998 Volume 273, Issue 42 Pages 27438-27448
Wei Wang, David K. Smith, Keith Moulding, and Hueih Min Chen

Abstract: A natural antibacterial peptide, cecropin B (CB), and designed analogs, CB-1 and CB-3, were synthesized. The three peptides have different structural characteristics, with CB having one hydrophobic and one amphipathic α-helix, CB-1 having two amphipathic α-helices, and CB-3 having two hydrophobic α-helices. These differences were used as the rationale for a study of their efficacy in breaking liposomes with different combinations of phosphatidic acid and phosphatidylcholine. Biosensor binding measurements and encapsulating dye leakage studies showed that the higher binding affinity of CB and CB-1 to the polar heads of lipids is not necessary for the peptides to be more effective at lysing lipid bilayers, especially when liposomes have a higher phosphatidic acid content. Kinetic studies, by intrinsic and extrinsic fluorescence stopped-flow measurements, revealed two transitional steps in liposome breakage by CB and CB-1, although only one kinetic step was found for CB-3. Circular dichroism stopped-flow measurements, monitoring the formation of secondary structure in the peptides, found one kinetic step for the interaction of all of the peptides with the liposomes. Also, the α-helical motif of the peptides was maintained after interacting with the liposomes. Based on these results, the mechanisms of liposome lysis by CB, CB-1, and CB-3 are discussed.
Protein, folding CD Stopped-flow Kinetic

"Effects Of Mutations In The γ-phosphate Binding Site Of Myosin On Its Motor Function"
J. Biol. Chem. 1998 Volume 273, Issue 42 Pages 27404-27411
Xiang-dong Li, Troy E. Rhodes, Reiko Ikebe, Taketoshi Kambara, Howard D. White,and Mitsuo Ikebe

Abstract: The role of the highly conserved residues in the γ-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.
Myosin Protein, folding

"The Proofreading Pathway Of Bacteriophage T4 DNA Polymerase"
J. Biol. Chem. 1998 Volume 273, Issue 36 Pages 22969-22976
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G E9, Canada

Abstract: The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. Mutator DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.
2-Aminopurine Fluorescence Stopped-flow Kinetic Complexation

"Developmental Changes In The Glycosylation Of Glycoprotein Hormone Free α Subunit During Pregnancy"
J. Biol. Chem. 1998 Volume 273, Issue 20 Pages 12068-12076
Martin Nemansky, N. Rao Thotakura, Curtis D. Lyons, Song Ye, Bruce B. Reinhold, Vernon N. Reinhold, and Diana L. Blithe

Abstract: Glycoprotein hormone α subunit, in its free form (free α), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 1[right-arrow]4/1[right-arrow]3- toward predominantly 1[right-arrow]6/1[right-arrow]6-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and α6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.

"Structural Analyses And Dynamics Of Soluble And Cell Wall-bound Phenolics In A Broad Spectrum Resistance To The Powdery Mildew Fungus In Barley"
J. Biol. Chem. 1998 Volume 273, Issue 15 Pages 9013-9022
Edda von R&ouml;penack, Adrian Parr, and Paul Schulze-Lefert

Abstract: High pressure liquid chromatography profiles of barley leaf epidermal soluble and cell wall-bound phenolics were analyzed in response to challenge with the fungal pathogen Erysiphe graminis f. sp. hordei. Only one soluble phenolic was found to accumulate differentially in a broad spectrum resistance reaction controlled by mlo resistance alleles in comparison to susceptible near isogenic Mlo lines. Structural analysis identified this compound as a novel phenolic conjugate, p-coumaroyl-hydroxyagmatine (p-CHA). p-CHA but not the nonhydroxylated derivative p-coumaroylagmatine exhibited antifungal activity both in vitro and in vivo. The accumulation of p-CHA in epidermal tissue correlated tightly with fungal penetration attempts of attacked host cells. Furthermore, upon penetration, epidermal cell wall-bound phenolics became resistant to saponification at sites of attempted fungal ingress (papilla), indicating a change in, or the addition of, different chemical bonding types. The switch in saponification sensitivity occurred at least 2 h earlier in the mlo-incompatible than in the Mlo-compatible interaction. Our results suggest that p-CHA and the speed of papillae compaction play important roles in non-race-specific powdery mildew defense.

"Molecular Cloning Of Human GDP-mannose 4,6-Dehydratase And Reconstitution Of GDP-fucose Biosynthesis In Vitro"
J. Biol. Chem. 1998 Volume 273, Issue 14 Pages 8193-8202
Francis X. Sullivan, Ravindra Kumar, Ronald Kriz, Mark Stahl, Guang-Yi Xu, Jason Rouse, Xiao-jia Chang, Amechand Boodhoo, Barry Potvin, and Dale A. Cumming

Abstract: We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.

"Reaction Of Prostaglandin Endoperoxide Synthase With Cis,cis-eicosa-11,14-dienoic Acid"
J. Biol. Chem. 1998 Volume 273, Issue 11 Pages 6046-6049
Vasilij Koshkin and H. Brian Dunford

Abstract: The pre-steady-state phase of the oxygenase reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11, 14-dienoic acid has been studied using stopped-flow techniques. Because some intermediate forms of prostaglandin endoperoxide synthase are spectrally indistinguishable, the enzyme and substrate transformations were monitored in parallel to simplify the interpretation of the kinetics. Over a wide range of conditions, the formation of the enzyme intermediate II, the form of compound I containing the tyrosyl radical, precedes substrate oxidation. This result supports the occurrence of a unimolecular conversion of compound I into intermediate II. Furthermore, the rate of intermediate II formation was stimulated by increased concentration of dienoic acid, perhaps because of increased occupation of the fatty acid binding site. The importance of the unimolecular formation of intermediate II was confirmed by simulated kinetics of the oxygenase reaction. These results provide evidence that intermediate II is the primary oxidant in the reaction of prostaglandin synthase with the dienoic acid, as it is with arachidonic acid.
cis-11,14-Eicosadienoic acid Kinetic Stopped-flow

"Characterization Of The Two-step Pathway For Inhibition Of Thrombin By α-ketoamide Transition State Analogs"
J. Biol. Chem. 1998 Volume 273, Issue 9 Pages 4843-4854
Sidney D. Lewis, Bobby J. Lucas, Stephen F. Brady, John T. Sisko, Kellie J. Cutrona, Philip E. J. Sanderson, Roger M. Freidinger, Shi-Shan Mao, Stephen J. Gardell and Jules A. Shafer

Abstract: The interaction of thrombin with several potent and selective α-ketoamide transition state analogs was characterized. L-370, 518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (Ki = 90 pM) and selective (>10(4)-fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed that k-1, the rate constant for dissociation of EI1, was comparable to k2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s-1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nM) largely due to an increase in k-1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1' N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K-1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pM) than L-370,518 due to its 16-fold higher k2 and 10-fold lower k-2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K-1 value for its α-ketoamide analog, L-370,518, were similar (5 and 14 nM, respectively). Likewise, the Ki value for L-372,011 and the K-1 value for its α-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the α-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the α-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.
Thrombin Kinetic Stopped-flow

"Involvement Of The C-terminal Domain Of An ATP-binding Subunit In The Regulation Of The ABC-type Nitrate/Nitrite Transporter Of The Cyanobacterium Synechococcus Sp. Strain PCC 7942"
J. Biol. Chem. 1997 Volume 272, Issue 43 Pages 27197-27201
Masaki Kobayashi, Roc&iacute;o Rodr&iacute;guez, Catalina Lara, and Tatsuo Omata

Abstract: In Synechococcus sp. strain PCC 7942, an ATP-binding cassette transporter encoded by the genes nrtA, nrtB, nrtC, and nrtD mediates active transport of nitrate and nitrite, which is inhibited by ammonium, a preferred source of nitrogen for the cyanobacterium. One of the ATP-binding subunits of the transporter, NrtC, has a distinct C-terminal domain of 380 amino acid residues. A mutant NC2, constructed by removal of this domain using genetic engineering techniques, assimilated low concentrations of nitrate and nitrite and accumulated nitrate intracellularly, showing that the domain is not essential for the transporter activities. Assimilation of low concentrations of nitrite was only partially inhibited by ammonium in NC2 but was completely inhibited in the wild-type cells. Cells of NC2 and its derivative (nitrate reductase-less strain NC4) carrying the truncated NrtC but not the cells with the wild-type NrtC accumulated nitrate intracellularly in the presence of ammonium in medium. These findings indicated that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the nitrate/nitrite transporter. In the presence of ammonium, NC2 could not assimilate nitrate despite its ability to accumulate nitrate intracellularly, which suggested that reduction of intracellular nitrate by nitrate reductase is also subject to inhibition by ammonium.

"Equilibrium And Kinetic Study Of The Conformational Transition Toward The Active State Of P21Ha-ras, Induced By The Binding Of BeF3- To The GDP-bound State, In The Absence Of GTPase-activating Proteins"
J. Biol. Chem. 1997 Volume 272, Issue 37 Pages 23138-23143
Jose Fernando D&iacute;az, Alain Sillen, and Yves Engelborghs

Abstract: Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-ras) to demonstrate that BeF3- binds to the GDPbullp21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25°C = 8.1±0.3 mM) and is endothermic (DeltaH = 22.3±1.6 kJ mol-1). The similarity between the GTP-bound form and the GDPbullBeF3- bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15±2 kJ mol-1 and DeltaH2 = 8±2 kJ mol-1). The kinetically determined enthalpy change of 23±4 kJ mol-1 is in excellent agreement with the equilibrium analysis. Flow injection analyis is used.
Protein, binding Fluorescence Kinetic Method comparison

"Anchor Structure Of Staphylococcal Surface Proteins. A BRANCHED PEPTIDE THAT LINKS THE CARBOXYL TERMINUS OF PROTEINS TO THE CELL WALL"
J. Biol. Chem. 1997 Volume 272, Issue 35 Pages 22285-22292
Hung Ton-That, Kym F. Faull, and Olaf Schneewind

Abstract: Surface proteins of Staphylococcus aureus are anchored to the cell wall by a mechanism requiring a COOH-terminal sorting signal. Previous work demonstrated that the sorting signal is cleaved at the conserved LPXTG motif and that the carboxyl of threonine (T) is linked to the staphylococcal cell wall. By employing different cell wall lytic enzymes, surface proteins were released from the staphylococcal peptidoglycan and their COOH-terminal anchor structure was revealed by a combination of mass spectrometry and chemical analysis. The results demonstrate that surface proteins are linked to a branched peptide (NH2-Ala-γ-Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the pentaglycine cross-bridge that is attached to the [epsilon]-amino of lysyl. This branched anchor peptide is amide-linked to the carboxyl of N-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.

"Structural Identification By Mass Spectrometry Of Oxidized Phospholipids In Minimally Oxidized Low Density Lipoprotein That Induce Monocyte/Endothelial Interactions And Evidence For Their Presence In Vivo"
J. Biol. Chem. 1997 Volume 272, Issue 21 Pages 13597-13607
Andrew D. Watson, Norbert Leitinger, Mohamad Navab, Kym F. Faull, Sohvi H&ouml;rkk&ouml;, Joseph L. Witztum, Wulf Palinski, Dawn Schwenke, Robert G. Salomon, Wei Sha, Ganesamoorthy Subbanagounder, Alan M. Fogelman, and Judith A. Berliner

Abstract: Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjunction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

"Control Of Activity Through Oxidative Modification At The Conserved Residue Cys66 Of Aryl Sulfotransferase IV"
J. Biol. Chem. 1997 Volume 272, Issue 14 Pages 9153-9160
A. David Marshall, John F. Darbyshire, Ann P. Hunter, Peter McPhie, and William B. Jakoby

Abstract: Oxidation at Cys66 of rat liver aryl sulfotransferase IV alters the enzymes catalytic activity, pH optima and substrate specificity. Although this is a cytosolic detoxification enzyme, the pH optimum for the standard assay substrate 4-nitrophenol is at pH 5.5; upon oxidation, the optimum changes to the physiological pH range. The principal effect of the change in pH optimum is activation, which is manifest by an increase in K[']cat without any major influence on substrate binding. In contrast, with tyrosine methyl ester as a substrate, the enzyme's optimum activity occurs at pH 8.0; upon oxidation, it ceases to be a substrate at any pH. The presence of Cys66 was essential for activation to occur, thereby providing a putative reason underlying the conserved nature of this cysteine throughout the phenol sulfotransferase family. Mapping of disulfides by mass spectrometry showed the critical event to be the oxidation of Cys66 to form a disulfide with either Cys232 or glutathione, either one is effective. These results point to a mechanism for regulating the activity of a key enzyme in xenobiotic detoxification during cellular oxidative stress.

"Effects Of External Osmotic Pressure On Vesicular Secretion From Bovine Adrenal Medullary Cells"
J. Biol. Chem. 1997 Volume 272, Issue 13 Pages 8325-8331
Ricardo Borges, Eric R. Travis, Spencer E. Hochstetler, and R. Mark Wightman

Abstract: Secretion of catecholamines from individual vesicles of bovine adrenal medullary cells was studied with amperometry in media of various osmolarities and compared with results obtained in isotonic physiological buffer (315 mosM). Hypotonic solutions caused an increase in the number of amperometric spikes evoked by brief exposure to 5 mM Ba2+. Under moderate hypertonic conditions (630 mosM), individual vesicular events were decreased in frequency, and lower amounts were secreted per event. Furthermore, the events were temporally broadened relative to those observed during release in isotonic conditions. At 970 mosM, exposure to 5 mM Ba2+ evoked even smaller secretory events that resemble the prespike feature that has been attributed to the initial opening of the fusion pore. The lack of large spikes is not due to failure of Ba2+ entry because fura-2 fluorescence reveals an increase in intracellular divalent ions. After exposure to Ba2+ in hypertonic solution, spikes could be induced with isotonic solution transiently directed onto the cell, but this process was not accompanied by a change in the concentration of intracellular divalent ions. Thus, this procedure provides an unique opportunity to temporally separate exocytotic secretion from entry of divalent ions.

"Substrate-binding Lipoprotein Of The Cyanobacterium Synechococcus Sp. Strain PCC 7942Involved In The Transport Of Nitrate And Nitrite"
J. Biol. Chem. 1997 Volume 272, Issue 5 Pages 3036-3041
Shin-ichi Maeda and Tatsuo Omata

Abstract: Of the four genes (nrtABCD) required for active transport of nitrate in the cyanobacterium Synechococcus sp. strain PCC 7942, nrtBCD encode membrane components of an ATP-binding cassette transporter involved in the transport of nitrite as well as of nitrate, whereas nrtA encodes a 45-kDa cytoplasmic membrane protein, the biochemical function of which remains unclear. Characterization of the nrtA deletional mutants showed that the 45-kDa protein is essential for the functioning of the nitrate/nitrite transporter. A truncated NrtA protein lacking the N-terminal 81 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, was shown to bind nitrate and nitrite with high affinity (Kd = 0.3 µM). Immunoblotting analysis using the antibody against the 45-kDa protein revealed a 48-kDa precursor of the protein, which accumulated in the cyanobacterial cells treated with globomycin, an antibiotic that specifically inhibits cleavage of the signal peptide of lipoprotein precursors. These findings indicated that the nrtA gene product is a nitrate- and nitrite-binding lipoprotein. The N-terminal sequences of putative cyanobacterial substrate-binding proteins suggested that lipoprotein modification of substrate-binding proteins of ATP-binding cassette transporters is common in cyanobacteria.

"Haloenol Lactone Is A New Isozyme-selective And Active Site-directed Inactivator Of Glutathione S-Transferase"
J. Biol. Chem. 1996 Volume 271, Issue 34 Pages 20421-20425
Jiang Zheng, Alyson E. Mitchell, A. Daniel Jones, and Bruce D. Hammock

Abstract: A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 µM) with murine Alpha, Mu, or Pi GST isozyme (1.0 µM) at pH 6.5, 37°C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, ~ 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37°C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 µM) was incubated with GST Pi isozyme (1.0 µM) in the presence of the competitive inhibitor S-hexylglutathione (10 µM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.

"Functional Organization Of Saposin C"
J. Biol. Chem. 1996 Volume 271, Issue 12 Pages 6874-6880
Xiaoyang Qi, Wen Qin, Ying Sun, Keiji Kondoh, and Gregory A. Grabowski

Abstract: Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid β-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid β-glucosidase activation properties to the carboxyl- and NH[(2)]-terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid β-glucosidase.

"Mechanism Of Soluble Epoxide Hydrolase"
J. Biol. Chem. 1995 Volume 270, Issue 45 Pages 26923-26930
Babak Borhan, A. Daniel Jones, Franck Pinot, David F. Grant, Mark J. Kurth, and Bruce D. Hammock

Abstract: [^18]O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H[(2)][^18]O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of [^18]O. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an α-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.

"Serpin-protease Complexes Are Trapped As Stable Acyl-enzyme Intermediates"
J. Biol. Chem. 1995 Volume 270, Issue 43 Pages 25309-25312
Daniel A. Lawrence, David Ginsburg, Duane E. Day, Mitchell B. Berkenpas, Ingrid M. Verhamme, Jan-Olov Kvassman and Joseph D. Shore

Abstract: The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped in either a tight Michaelis- like structure, a tetrahedral intermediate, or an acyl-enzyme. In this report we show that, upon association with a target protease, the serpin reactive-center loop (RCL) is cleaved resulting in formation of an acyl-enzyme intermediate. This cleavage is coupled to rapid movement of the RCL into the body of the protein bringing the inhibitor closer to its lowest free energy state. From these data we suggest a model for serpin action in which the drive toward the lowest free energy state results in trapping of the protease-inhibitor complex as an acyl-enzyme intermediate. (FIA was used!)
Enzyme, activity Fluorescence Kinetic

"Participation Of Acetylpseudouridine In The Synthesis Of A Peptide Bond In Vitro"
J. Biol. Chem. 1995 Volume 270, Issue 36 Pages 21040-21044
D. Denise Wood, Henrianna Pang, Andrew Hempel, Norman Camerman, Byron G. Lane, and Mario A. Moscarello

Abstract: Uracil, uridine, and pseudouridine were acetylated by refluxing in acetic anhydride, and the products of acetylation were incubated with a synthetic peptide(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) that corresponds to the N-terminal 21 amino acid residues of human myelin basic protein. Peptide bond formation, at the Nα terminus in peptide 1-21, was obtained with acetyluracil and acetylpseudouridine, but not with acetyluridine. Transfer of an acetyl group from acetyluracil and acetylpseudouridine depended on acetylation in the N-heterocycle. X-ray crystallographic analysis definitively established N-1 as the site of acetylation in acetyluracil. Mass spectrometry of the acetylation products showed that one acetyl group was transferred to peptide 1-21, in water, by either acetyluracil or acetylpseudouridine at pH~6. Release of the acetyl group by acylaminopeptidase regenerated peptide 1-21 (mass spectrometry) and automated sequencing (for five cycles) of the regenerated (deacetylated) peptide demonstrated that the N terminus was intact. The findings are discussed in the context of a possible role for pseudouridine in ribosome-catalyzed peptidyltransfer, with particular reference being made to similarities between the possible mechanism of acyl transfer by acetyluracil/pseudouridine and the mechanism of carboxyl transfer by carboxylbiotin in acetyl CoA carboxylase. The possibility that idiosyncratic appearance of a wide range of acyl substituents in myelin basic protein could be related to a peculiar involvement of ribosomal pseudouridine is mentioned.

"Isoleukotrienes Are Biologically Active Free Radical Products Of Lipid Peroxidation"
J. Biol. Chem. 1995 Volume 270, Issue 29 Pages 17273-17278
Kathleen A. Harrison and Robert C. Murphy

Abstract: The free radical oxidation of arachidonic acid esterified to glycerophospholipids is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins derived from prostaglandin H2 synthase. Furthermore, certain isoprostanes have been found to exert biological activity through endogenous receptors on cell surfaces. Using mass spectrometry and ancillary techniques, the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine was studied in the search for products of arachidonic acid isomeric to the leukotrienes that are derived from 5-lipoxygenase-catalyzed metabolism of arachidonic acid. Several conjugated triene metabolites were chromatographically separated from known 5-lipoxygenase products and structures characterized as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid esterified to the glycerophosphocholine backbone. We have termed these products as B4-isoleukotrienes. Following saponification some, but not all, B4-isoleukotrienes were found to exert biological activity in elevating intracellular calcium in Indo-1-loaded human polymorphonuclear leukocytes. This activity could be blocked by a leukotriene B4 receptor antagonist. An EC50 of approximately 30 nM was determined for one unique B4-isoleukotriene with a relative retention index of 2.54. We have shown that free radical processes can lead to the formation of biologically active isoleukotrienes in glycerophosphocholine liposomes, and we propose that B4-isoleukotrienes may also be formed in membrane glycerophospholipids as a result of lipid peroxidation during tissue injury. Such B4-isoleukotrienes could then mediate events of tissue damage through activation of leukotriene B4 receptors on target cells.

"Transient Free Radicals In Iron/oxygen Reconstitution Of Mutant Protein R2 Y122F. Possible Participants In Electron Transfer Chains In Ribonucleotide Reductase"
J. Biol. Chem. 1995 Volume 270, Issue 21 Pages 12361-12372
Margareta Sahlin, Gunter Lassmann, Stephan Potsch, Britt-Marie Sjoberg, and Astrid Graslund

Abstract: Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein Y122F leads to formation of a diferric center similar to that of the wild- type R2 protein of Escherichia coli ribonucleotide reductase. This reconstitution reaction requires two extra electrons, supplied or transferred by the protein matrix of R2. We observed several transient free radical species using stopped-flow and freeze quench EPR and stopped-flow UV-visible spectroscopy. Three of the radicals occur in the time window 0.1-2 s, i.e. concomitant with formation of the diferric site. They include a strongly iron-coupled radical (singlet EPR signal) observed only at < or = 77 K, a singlet EPR signal observed only at room temperature, and a radical at Tyr-356 (light absorption at 410 nm), an invariant residue proposed to be part of an electron transfer chain in catalysis. Three additional transient radicals species are observed in the time window 6 s to 20 min. Two of these are conclusively identified, by specific deuteration, as tryptophan radicals. Comparing side chain geometry and distance to the iron center with EPR characteristics of the radicals, we propose certain Trp residues in R2 as likely to harbor these transient radicals.
Spectrophotometry Spectroscopy Stopped-flow Kinetic

"Molecular And Biochemical Evidence For The Involvement Of The Asp-333-His-523 Pair In The Catalytic Mechanism Of Soluble Epoxide Hydrolase"
J. Biol. Chem. 1995 Volume 270, Issue 14 Pages 7968-7974
Franck Pinot, David F. Grant, Jeffrey K. Beetham, Anthony G. Parker, Babak Borhan, Steve Landt, Arthur D. Jones, and Bruce D. Hammock

Abstract: In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other α/β hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333→Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333→Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333→Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333→Asn mutant at 37°C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

"Correlation Of Real-time Catecholamine Release And Cytosolic Ca[IMAGE] At Single Bovine Chromaffin Cells"
J. Biol. Chem. 1995 Volume 270, Issue 10 Pages 5353-5359
Jennifer M. Finnegan and R. Mark Wightman

Abstract: Previous investigations of the role of Ca2+ in stimulus-secretion coupling have been undertaken in populations of adrenal chromaffin cells. In the present study, the simultaneous detection of intracellular Ca2+, with the fluorescent probe fura-2, and catecholamine release, using a carbon-fiber microelectrode, are examined at single chromaffin cells in culture. Results from classic depolarizing stimuli, high potassium (30-140 mM) and 1,1-dimethyl-4-phenylpiperazinium (3-50 µM), show a dependence of peak cytosolic Ca2+ concentration and catecholamine release on secretagogue concentration. Catecholamine release induced by transient high K+ stimulation increases logarithmically with K+ concentration. Continuous exposure to veratridine (50 µM) induces oscillations in intracellular Ca2+ and at higher concentrations (100 µM) concomitant fluctuation of cytosolic Ca2+ and catecholamine secretion. Mobilization of both caffeine and inositol trisphosphate-sensitive intracellular Ca2+ stores is found to elicit secretion with or without extracellular Ca2+. Caffeine-sensitive intracellular Ca2+ stores can be depleted, refilled, and cause exocytosis in medium without Ca2+. Single cell measurement of exocytosis and the increase in cytosolic Ca2+ induced by bradykinin-activated intracellular stores reveal cell to cell variability in exocytotic responses which is masked in populations of cells. Taken together, these results show that exocytosis of catecholamines can be induced by an increase in cytosolic Ca2+ either as a result of transmembrane entry or by release of internal stores.

"CD14: Physical Properties And Identification Of An Exposed Site That Is Protected By Lipopolysaccharide"
J. Biol. Chem. 1995 Volume 270, Issue 10 Pages 5213-5218
Michael D. McGinley, Linda O. Narhi, Michael J. Kelley, Elyse Davy, John Robinson, Michael F. Rohde, Samuel D. Wright, and Henri S. Lichenstein

Abstract: Soluble CD14 (sCD14) is a 55-kDa serum protein that binds lipopolysaccharide (LPS) and mediates LPSdependent responses in a variety of cells. Using recombinant sCD14 expressed in Chinese hamster ovary (CHO) cells, we examined the structural characteristics of sCD14 and sCD14[bullet]LPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial β-sheet (40%) and a well-defined tertiary structure with the tryptophan residues located in environments with different degrees of hydrophobicity and solvent exposure. The spectra of the sCD14[bullet]LPS complex are identical within experimental error to the uncomplexed sCD14. Changes in surface accessibility upon LPS binding were examined using limited proteolysis with endoproteinase Asp-N. This analysis revealed that aspartic acid residues at amino acids 57, 59, and 65 are susceptible to cleavage by Asp-N, while the same residues are protected from proteolytic cleavage in the sCD14[bullet]LPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14.

"Kinetics Of Bilirubin Transfer Between Serum Albumin And Membrane Vesicles"
J. Biol. Chem. 1995 Volume 270, Issue 3 Pages 1074-1081
Stephen D. Zucker, Wolfram Goessling, and John L. Gollan

Abstract: Unconjugated bilirubin is transported in the plasma bound primarily to serum albumin, from which it is taken up and metabolized by the liver. To better characterize the mechanism of bilirubin delivery to the hepatocyte, stopped-flow techniques were utilized to study the kinetics of bilirubin transfer between serum albumin and both model phospholipid and native hepatocyte plasma membrane vesicles. The transfer process was best described by a single exponential function, with rate constants of 0.93±0.04, 0.61 ±0.03, and 0.10±0.01 s-1 (±S.D.) at 25°C for human, rat, and bovine serum albumins, respectively. The observed variations in rate with respect to donor and acceptor concentrations provide strong evidence for the diffusional transfer of free bilirubin. Thermodynamic analysis suggests that the binding site on bovine serum albumin demonstrates higher specificity for the bilirubin molecule than that on human or rat serum albumin, which exhibit similar binding characteristics. Kinetic analysis of bilirubin transfer from rat serum albumin to isolated rat basolateral liver plasma membranes indicates that the delivery of albumin-bound bilirubin to the hepatocyte surface occurs via aqueous diffusion, rather than a collisional process, thereby mitigating against the presence of an 'albumin receptor'.

"A Membrane-mediated Catalytic Event In Prothrombin Activation"
J. Biol. Chem. 1994 Volume 269, Issue 41 Pages 25838-25848
Chun Kun, Eric Hayes, and Kenneth G. Mann

Abstract: Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.
Enzyme, prothrombinase Electrode Fluorescence Stopped-flow