University of North Florida
Browse the Citations
-OR-

Contact Info

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

View Stuart Chalk's profile on LinkedIn

Proceedings of the National Academy of Sciences

  • Publisher:
  • FAD Code: PNAS
  • CODEN: PNASA6
  • ISSN: 0027-8424
  • Abbreviation: Proc. Natl. Acad. Sci. USA
  • DOI Prefix: 10.1073/pnas
  • Language: English
  • Comments: Fulltext from 1915 V1

Citations 6

"Purification And Kinetic Analysis Of Recombinant CA XII, A Membrane Carbonic Anhydrase Overexpressed In Certain Cancers"
Proc. Natl. Acad. Sci. USA 2000 Volume 97, Issue 26 Pages 14212-14217
Barbara Ulmasov, Abdul Waheed, Gul N. Shah, Jeffrey H. Grubb, William S. Sly, Chingkuang Tu, and David N. Silverman

Abstract: Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO2 were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of O-18 between CO2 and water determined by mass spectrometry. The catalysis of CO2 hydration by soluble CA XII has a maximal value of k(cat)/K-cm at 34 muM-1.s-1, which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO2 hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO2/HCO3- homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.

"Improved Barley Broiler Feed With Transgenic Malt Containing Heat-stable (1,3-1,4)-β-glucanase"
Proc. Natl. Acad. Sci. USA 2000 Volume 97, Issue 25 Pages 13512-13517
Diter von Wettstein, Galina Mikhaylenko, John A. Froseth, and C. Gamini Kannangara

Abstract: The low nutritional value of barley for poultry is because of the absence of an intestinal enzyme for efficient depolymerization of (1,3-1,4)-β -glucan, the major polysaccharide of the endosperm cell walls. This leads to high viscosity in the intestine, limited nutrient uptake, decreased growth rate, and unhygienic sticky droppings adhering to chickens and floors of the production cages. Consequently, the 7.5 billion broiler chickens produced annually in the United States are primarily raised on corn-soybean diets. Here we show that addition to normal barley of 6.2% transgenic malt containing a thermotolerant (1,3-1,4)-β -glucanase (4.28 µg g-1 soluble protein) provides a weight gain equivalent to corn diets. The number of birds with adhering sticky droppings is drastically reduced. Intestines and excrements of chickens fed the barley control diet contained large amounts of soluble (1,3-1,4)-β-glucan, which was reduced by 75 and 50%. respectively, by adding transgenic malt to the diet. The amount of active recombinant enzyme in the small intestine corresponded to that present in the feed, whereas an 11-fold concentration of the enzyme was observed in the ceca, and a 7.5-fold concentration occurred in the excrement. Glycosylation of the β-glucanase isolated from the ceca testified to its origin from the transgenic barley. Analysis of the data from this trial demonstrates the possibility of introducing individual recombinant enzymes into Various parts of the gastrointestinal tract of chickens with transgenic malt and thereby the possibility of evaluating their effect on the metabolism of a given ingredient targeted by the enzyme.
Viscosity

"Mass Spectrometry And Immobilized Enzymes For The Screening Of Inhibitor Libraries"
Proc. Natl. Acad. Sci. USA 2000 Volume 97, Issue 22 Pages 12008-12013
Mark T. Cancilla, Michael D. Leavell, Jason Chow, and Julie A. Leary

Abstract: A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis, Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening,of additional new libraries without adding additional enzyme.

"Highly Water-permeable Type I Alveolar Epithelial Cells Confer High Water Permeability Between The Airspace And Vasculature In Rat Lung"
Proc. Natl. Acad. Sci. USA 1998 Volume 95, Issue 6 Pages 2991-2996
Leland G. Dobbs*, Robert Gonzalez, Michael A. Matthay, Ethan P. Carter, Lennell Allen, and A. S. Verkman

Abstract: Water permeability measured between the airspace and vasculature in intact sheep and mouse lungs is high. More than 95% of the internal surface area of the lung is lined by alveolar epithelial type I cells. The purpose of this study was to test whether osmotic water permeability (Pf) in type I alveolar epithelial cells is high enough to account for the high Pf of the intact lung. Pf measured between the airspace and vasculature in the perfused fluid-filled rat lung by the pleural surface fluorescence method was high (0.019±0.004 cm/s at 12°C) and weakly temperature-dependent (activation energy 3.7 kcal/mol). To resolve the contributions of type I and type II alveolar epithelial cells to lung water permeability, Pf was measured by stopped-flow light scattering in suspensions of purified type I or type II cells obtained by immunoaffinity procedures. In response to a sudden change in external solution osmolality from 300 to 600 mOsm, the volume of type I cells decreased rapidly with a half-time (t1/2) of 60-80 ms at 10°C, giving a plasma membrane Pf of 0.06-0.08 cm/s. Pf in type I cells was independent of osmotic gradient size and was weakly temperature-dependent (activation energy 3.4 kcal/mol). In contrast, t1/2 for type II cells in suspension was much slower, approximately 1 s; Pf for type II cells was 0.013 cm/s. Vesicles derived from type I cells also had a very high Pf of 0.06-0.08 cm/s at 10°C that was inhibited 95% by HgCl2. The Pf in type I cells is the highest measured for any mammalian cell membrane and would account for the high water permeability of the lung.
Cells Water Nephelometry Stopped-flow

"RecA Tests Homology At Both Pairing And Strand Exchange"
Proc. Natl. Acad. Sci. USA 1997 Volume 94, Issue 22 Pages 11863-11868
L. ROCHELLE BAZEMORE, EWA FOLTA-STOGNIEW, MASAYUKI TAKAHASHI, AND CHARLES M. RADDING

Abstract: RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases. Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences. The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates. In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches. The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14%. These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange. Flow Injection Analysis is used.
Protein, activity Bacteria Fluorescence Kinetic

"Native-like Structure Of A Protein-folding Intermediate Bound To The Chaperonin GroEL"
Proc. Natl. Acad. Sci. USA 1997 Volume 94, Issue 4 Pages 1080-1085
Matthew S. Goldberg, Jing Zhang, Stacey Sondek, C. Robert Matthews, Robert O. Fox, and Arthur L. Horwich

Abstract: The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel β-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL. Flow injection analysis was used.
Protein Fluorescence Stopped-flow