Determination of the kinetic parameters for phospholipase C (Bacillus cereus) on different phospholipid substrates using a chromogenic assay based on the quantitation of inorganic phosphate

The kinetic parameters of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) have been evaluated for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine substrates with a new assay based on the quantitation of inorganic phosphate (Pi). Treatment of the phosphomonoester product of the PLC(Bc)-catalyzed hydrolysis of these phospholipids with alkaline phosphatase releases Pi. This Pi forms a complex with ammonium molybdate that is then reduced by ascorbic acid to provide a blue molybdenum chromogen with an absorbance maximum at 700 nm. This highly sensitive assay may be used to determine accurately less than 5 nmol of Pi in solution. Performing the assay in 96-well plates provides a rapid and convenient method to evaluate a variety of phospholipids as substrates for PLC(Bc). The assay has been utilized to ascertain the kinetic constants for the PLC(Bc)-catalyzed hydrolysis of 1,2-dihexanoyl-sn-glycero- 3-phosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, and 1,2- dihexanoyl-sn-glycero-3-phospho-L-serine. It is found that these compounds are substrates for the enzyme with their V(max)s being in the order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine.