Purification and characterization of platelet-activating factor acetylhydrolase II from bovine liver cytosol.
Platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF by removing the acetyl group at the sn-2 position, is distributed widely in plasma and tissues. In a previous study, we demonstrated that the PAF acetylhydrolase activity present in the soluble fraction of bovine brain cortex could be separated chromatographically into three peaks (tentatively designated isoforms Ia, Ib, and II) (Hattori, M., Arai, H., and Inoue, K. (1993) J. Biol. Chem. 268, 18748-18753). In this study, these three isoforms were also detected in kidney and liver cytosols, although their relative activity ratios in these tissues differed. In particular, isoform II was responsible for the majority of the bovine liver PAF acetylhydrolase activity. We purified isoform II from bovine liver cytosol to near homogeneity and demonstrated that it is a single 40-kDa polypeptide. This enzyme was inactivated by diisopropyl fluorophosphate and 5,5'-dithiobis(2-nitrobenzoic acid), suggesting that both serine and cysteine residues are required for the enzyme activity, and [3H]diisopropyl fluorophosphate labeled only the 40-kDa polypeptide, confirming the enzyme's identity. Isoform II showed a comparatively broader substrate specificity than isoform Ib. Isoform II hydrolyzed propionyl and butyroyl moieties at the sn-2 position approximately half as effectively as it did PAF, whereas isoform Ib hardly hydrolyzed these substrates. Taken together with previous data, the current findings indicate that tissue cytosol contains at least two types of PAF acetylhydrolase with respect to polypeptide composition, substrate specificity, and tissue distribution and suggest that these two enzymes may share distinct physiological functions in tissues.
Hattori
K
K
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hattori
M
M
Adachi
H
H
Tsujimoto
M
M
Arai
H
H
Inoue
K
K
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D007527:Isoenzymes / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D010972:Platelet Activating Factor; D012694:Serine; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase