Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay

The substrate specificity for phospholipase D from Streptomyces chromofuscus (PLD(Sc)) has been determined utilizing an assay based on the quantitation of inorganic phosphate. 1,2-Di-n-hexanoyl phosphatidylcholine (C6PC), phosphatidylethanolamine (C6PE), phosphatidylserine (C6PS), phosphatidylglycerol (C6PG), and an unnatural phospholipid bearing a neohexyl headgroup (C6PDB) were examined as substrates. The assay relies on the quenching of the PLD(Sc)-catalyzed hydrolysis of the phospholipid substrates with EDTA followed by the hydrolysis of the phosphatidic acid product with alkaline phosphatase. The inorganic phosphate thus released is quantitated through the formation of a complex with ammonium molybdate, which has an absorbance maximum at 700 nm. To minimize the time involved and the reagents consumed, the assay is conducted in 96-well plates. The results of this study indicate that the catalytic efficiency for PLD(Sc) on the substrates is C6PC >> C6PS ≃ C6PE > C6PG >> C6PDB. (C) 2000 Academic Press.