Phospholipase A2 from bovine seminal plasma is a platelet-activating factor acetylhydrolase.
The major phospholipase A2 activity from bovine seminal plasma was recently purified [Soubeyrand, Khadir, Brindle and Manjunath (1997) J. Biol. Chem. 272, 222-227]. We here show that the 60 kDa enzyme is in fact a platelet-activating factor acetylhydrolase (PAF-AH). Sequences of the N-terminus as well as of internal fragments showed 100% identity with the cDNA-deduced sequences of bovine plasma PAF-AH. The enzyme has kinetic properties similar to those of the human serum PAF-AH. Although capable of hydrolysing long-chained phosphatidylcholine, it displayed a highly preferential activity towards PAF. The enzyme activity towards phosphatidylcholine, but not PAF, was Ca2+-dependent. Biochemical characterization revealed that the enzyme is extensively N-glycosylated and that it exists predominantly as a dimer in solution. Western blot analysis revealed that the enzyme is highly heterogeneous in charge, with a maximal distribution at an isoelectric point of approx. 5.7. The enzyme was expressed exclusively in the seminal vesicles and the ampulla. No association of the enzyme with either epididymal or ejaculated spermatozoa could be detected.
Soubeyrand
S
S
Guy-Bernier Research Centre, 5415 Boulevard de L'Assomption, Montreal, Qu-ebec, Canada H1T 2M4.
Lazure
C
C
Manjunath
P
P
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012661:Semen / Q000201:enzymology*; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D013388:Succinimides / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010972:Platelet Activating Factor; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D010741:Phospholipases A; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium