Abstract: Phospholipase D was assayed by FIA (schematic diagram given) with, as substrate, liposomes prepared from phosphatidylcholine. The choline produced is converted in the presence of choline oxidase into betaine and H2O2 which was detected by Co(II)-catalyzed luminol chemiluminescence. A differential experimental set-up (diagram given) was used to study kinetic parameters of phospholipase D from Streptomycesc hromofuscus, Actinomadura spp. and Brassica oleratia. Calibration graphs were linear from 1-100 miu/ml. The detection limit was 75 pmol choline released/min which compared favourably with detection limits by amperometric (0.3 nmol), titrimetric (20 nmol) and spectrophotometric (50 nmol) detection methods. A precisely working automated system for the investigation of phospholipases D (PLDs, EC 3.1.4.4) from plant and microbial sources with flow injection analysis (FIA) has been developed. The two versions of the FIA setup described are based on the oxidation of choline liberated from phosphatidylcholine by PLD action and catalyzed by choline oxidase and the chemiluminescence detection of hydrogen peroxide produced by this reaction. The correlation between this chemiluminescence signal and the PLD activity was linear in the range between 1 and 100 mU/ml PLD. The sampling frequency was 12 samples per hour. This method was used to compare three different PLDs from cabbage and microbial sources with respect to their pH optima, temperature stability, effectors, and v/[S]-characteristics.

Abstract: The activity of phospholipase D (PLD) from Streptomyces chromofuscus in two-phase systems consisting of a PLD containing aqueous phase and a phosphatidylcholine (PC) containing organic solvent phase was investigated. Two-phase systems with highly reproducible segmentation were generated in a continuous-flow system using a capillary segmentor. Between 0.01 and 2 U/mL PLD from Streptomyces chromofuscus were detected in a water/hexane two-phase system. The linear detection range was 0.01 to 0.5 U PLD/mL. The apparent KM of Streptomyces chromofuscus PLD in a water/hexane system was 41.43 µM PC, the apparent Vmax was 3.68 µmol min-1 mg-1. The variation of PLD activity in different organic solvents correlated with the solvent's hydrophobicity. If anionic detergents or primary alcohols were added to the two-phase system, concentration dependent activation of the PLD was observed

Abstract: A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1 x 10^-7 mol/L, with rsd 1.8 to 2.8% in the range 2-10 x 10^-5 mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage.