Conductimetric Assays for the Hydrolase and Transferase Activities of Phospholipase D Enzymes

Abstract

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.