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 (PLCBc) 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 PLCBc-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 PLCBc. The assay has been utilized to ascertain the kinetic constants for the PLCBc-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 VmaxS being in the order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine.
Hergenrother
P J
PJ
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Martin
S F
SF
D001412:Bacillus subtilis / Q000201:enzymology*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002863:Chromogenic Compounds; D007700:Kinetics; D008982:Molybdenum; D010710:Phosphates / Q000032:analysis; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000032:analysis* / Q000378:metabolism*
D002863:Chromogenic Compounds; D010710:Phosphates; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; D008982:Molybdenum; D010738:Type C Phospholipases