Purification and characterization of bovine brain platelet-activating factor acetylhydrolase.
Platelet-activating factor (PAF) acetylhydrolase, which removes the acetyl moiety at the sn-2 position, has been found in plasma and tissue cytosol. PAF acetylhydrolase in bovine brain cytosol was chromatographically separated into three distinct fractions, all of which exhibited pH optima in the neutral to mild alkaline region and were unaffected by EDTA. We have purified the major fraction of the enzyme to near homogeneity. The purified enzyme had a molecular mass of about 100 kDa, as estimated by gel filtration chromatography, and gave three distinct bands of 45, 30, and 29 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides exclusively co-migrated with the activity throughout the purification steps. These data suggest that this set of polypeptides corresponds to the subunits of bovine brain PAF acetylhydrolase. Diisopropyl fluorophosphate completely inhibited the activity at 0.1 mM. [3H]Diisopropyl fluorophosphate labeled only the 29-kDa polypeptide, suggesting that this polypeptide possesses an active serine residue(s). The purified enzyme displayed similar activity against PAF and oxidatively modified phosphatidylcholine, but did not hydrolyze phosphatidylcholine or phosphatidylethanolamine with two long chain acyl groups. Thus, the intracellular PAF acetylhydrolase is likely to be a new member of the calcium-independent phospholipases A2 in mammalian tissues.
Hattori
M
M
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Arai
H
H
Inoue
K
K
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D007531:Isoflurophate; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase