Chromogenic assay for phospholipase D from Streptomyces chromofuscus: application to the evaluation of substrate analogs.

A rapid and convenient chromogenic assay for phospholipase D from Streptomyces chromofuscus (PLDSc) has been developed that converts the choline generated from the enzyme-catalyzed hydrolysis of phospholipids into a chromogenic dye. By quenching the reaction with EDTA at defined times, an initial rate curve is produced from which a kcat and K(m) can be readily derived. This assay has been applied to the biological evaluation of several substrate analogs, all of which appear to be activators rather than substrates or inhibitors of this enzyme. Performing the assay in 96-well microtiter plates allows for the easy screening of potential effectors of this enzyme.