Purification and characterization of 45 kDa PAF acetylhydrolase from bovine colostrum.
Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetylhydrolase (PAF-AH) activity has been identified in bovine colostrum and high levels of this activity are found in early colostrum (within 24 h after parturition). In this study, PAF-AH in early colostrum was purified by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl 650M, DEAE-Sepharose, heparin-Sepharose, hydroxyapatite, chelating-Sepharose and Mono Q HPLC column chromatography. This enzyme is a monomeric polypeptide with a molecular weight of approximately 45 kDa on 12.5% SDS-PAGE. The V(max) and K(m) for PAF-AH were 87.6 microM and 7.96 nmol/min/mg respectively. This enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetamide and p-bromophenacylbromide, suggesting that both serine and histidine residues are required for enzyme activity. It was not inactivated by NaF or dithiothreitol. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. Accordingly, this enzyme is distinct from phospholipase A(2). In addition, PAF-AH selectively hydrolyzed oxidatively modified phosphatidylcholine. Furthermore, this enzyme was shown by Western blot analysis using antibody to human plasma PAF-AH to be plasma type PAF-AH. These results clearly demonstrate that 45 kDa plasma type PAF-AH activity exists in bovine colostrum.
Moon
Tae Chul
TC
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea.
Son
So Young
SY
Chang
Hyeun Wook
HW
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003126:Colostrum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity