Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat.
Peritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A2. The activity required Ca2+ ion and had a pH optimum of 9 (Chang, H.W., Kudo, I., Hara, S., Karasawa, K., & Inoue, K. (1986) J. Biochem. 100, 1099-1101). This phospholipase A2 was purified about 14,000-fold to near homogeneity by the sequential use of column chromatography on Sephadex G-75, Toyopearl HW-65, and TSK ODS-120T reverse-phase HPLC. The final preparation showed a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 13,500. The enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). When 1-acyl-2-[1-14C]inoleoyl-sn-glycero-3-phospholipids were used as a substrate, the apparent Km values were 0.027 mM with PE, 0.032 mM with PS, and 0.1 mM with PC, and the Vmax values were 105 mumol/min/mg with PE, 71 mumol/min/mg with PC. The enzyme activity was inhibited by p-bromophenacyl bromide, dithiothreitol, and mepacrine. The amino acid sequence of the NH2-terminal portion and the amino acid composition of the purified enzyme were determined. They were different from those of rat pancreatic phospholipase A2, but very similar to those of phospholipase A2 secreted from rat platelets.
Chang
H W
HW
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Kudo
I
I
Tomita
M
M
Inoue
K
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002364:Caseins / Q000494:pharmacology*; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010529:Peritoneal Cavity / Q000201:enzymology*; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D002364:Caseins; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2