Human plasma platelet-activating factor acetylhydrolase. Purification and properties.
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid synthesized by a variety of cell types upon appropriate stimulation. PAF is a potent hypotensive factor and it activates platelets and inflammatory cells at concentrations as low as 10(-10) M. Removal of the acetyl moiety at the sn-2 position abolishes the biological activity and this reaction is catalyzed by a specific acetylhydrolase present in plasma and animal tissues. Ultracentrifugation in density gradients showed that 30% of the activity is associated with high density lipoproteins and 70% with low density lipoproteins. We have purified the plasma low density lipoprotein-associated activity to near homogeneity using a rapid assay based on the separation of [3H]acetate from 1-O-alkyl-2-[3H]acetyl-sn-glycerol-3-phosphocholine on disposable reversed-phase columns. The enzyme was purified by 25,000-fold and approximately 10% of the starting activity was recovered. Plasma PAF-acetylhydrolase has an apparent molecular weight of 43,000, does not require calcium, has preference for micellar versus monomeric substrate, and exhibits surface dilution kinetics. The purified protein has an apparent Km of 13.7 microM and a Vmax of 568 mumol/h/mg with micellar PAF. It can act both on 1-O-alkyl and 1-acyl substrates and on ethanolamine analogs of PAF. However, the enzyme has a marked preference for the sn-2 acetyl residue and therefore can be considered as a specific PAF-acetylhydrolase.
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood; D008077:Lipoproteins, LDL / Q000097:blood; D008823:Micelles; D008970:Molecular Weight; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000097:blood*; D010972:Platelet Activating Factor / Q000378:metabolism; D011136:Polysorbates; D012995:Solubility; D013379:Substrate Specificity
D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008823:Micelles; D010972:Platelet Activating Factor; D011136:Polysorbates; D010740:Phospholipases; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase