title: Purification and characterization of lysophospholipase released from rat platelets.
abstract: Lysophospholipase released from rat platelets upon activation with thrombin has been purified to near homogeneity by sequential column chromatography on heparin-Sepharose, CM-Sephadex C-50, and TSK gel G2000SW. The final preparation showed a single band with a molecular mass of 32,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The purified enzyme was heat-labile and inactivated after 5 min at 60 degrees C. It showed a broad pH optimum (pH 6-10) and required a divalent cation, such as Ca2+, for the optimal activity. Appreciable activity, however, was observed in the presence of EDTA. Lysophospholipase activity was inhibited by diisopropylfluorophosphate and dithiothreitol. This enzyme activity was retained by a concanavalin A-Sepharose column and eluted with methyl-alpha-D-mannoside. Treatment of lysophospholipase with peptide: N-glycosidase F gave degraded products, suggesting that this protein contain N-linked carbohydrate chains. The purified enzyme was specific to 1-acyl-sn-glycero-3-phospho-L-serine; none of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, and 1-acyl-sn-glycero-3-phospho-D-serine was hydrolyzed appreciably.
authors.affiliation: Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
authors.forename: S
authors.initials: S
authors.lastname: Higashi
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: I
authors.initials: I
authors.lastname: Kudo
authors.forename: K
authors.initials: K
authors.lastname: Inoue
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D008245:Lysophospholipase / Q000032:analysis / Q000302:isolation & purification*; D008297:Male; D010740:Phospholipases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology
chemical_list: D010740:Phospholipases; D008245:Lysophospholipase; D013917:Thrombin