The platelet-activating factor acetylhydrolase from human erythrocytes. Purification and properties.
Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine) is a biologically active phospholipid. Tissues, blood cells, and plasma contain PAF acetylhydrolases (calcium independent phospholipase A2 activities) that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups. They inactivate PAF and thereby determine PAF accumulation. We purified the PAF acetylhydrolase from human erythrocytes 15,600-fold. The enzyme has a molecular weight of 25,000, it behaves as a dimer during gel filtration, and it is a previously uncharacterized cytosolic esterase, as it has a unique amino-terminal sequence. The erythrocyte PAF acetylhydrolase requires the addition of sulfhydryl agents for maximal activity, is inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), NaF, diisopropyl fluorophosphate, diethylpyrocarbonate, p-bromophenacylbromide, and a number of proteases. Antibodies against the purified protein precipitate all PAF hydrolase activity from erythrocyte lysates. The erythrocyte PAF acetylhydrolase is specific for short or oxidized sn-2 acyl residues. It exhibits surface dilution kinetics, suggesting that hydrolysis occurs at lipid interfaces. This suggests that this enzyme acts in vivo as a scavenger of oxidatively fragmented phospholipids that are toxic to the cell.
Stafforini
D M
DM
Nora Eccles Harrison Cardiovascular Research and Training Institute, Salt Lake City, Utah.
Rollins
E N
EN
Prescott
S M
SM
McIntyre
T M
TM
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D013379:Substrate Specificity
D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase