Abstract
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.
Highlights
PURIFICATION AND PROPERTIES*Blood cells, and plasma contain PAF acetylhydrolases (calcium independent phospholipase Az activities) that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups
From the Nora Eccles Harrison Cardiovascular Research and Training Institute, tEheccles Program in HumanMolecular Bio1og.y and Genetics, and the Departmentsof Internal Medicine and Biochemistry, Universityof Utah, Salt Lake City, Utah84112
Blood cells, and plasma contain PAF acetylhydrolases that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups
Summary
Blood cells, and plasma contain PAF acetylhydrolases (calcium independent phospholipase Az activities) that catalyze the hydrolysis of phospholipids containing short chain sn-2 acyl groups. They inactivate PAF and thereby determine PAF accumulation. 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. Wcheose humanerythrocytesasthestartingmaterialto purify an intracellular PAF acetylhydrolase because these cells can be isolated in large quantities and they have high levels of the column DEAE-step was placed on a Sephacryl S-300 column (2.5 X 90 cm)equilibratedin 10 mM sodium phosphate buffer (pH 6.8) containing 250 PM DTE and 1mM EDTA, at a flow rate of 19 ml/h. Phenyl-Sepharose Column-The preparation was placed on a phenyl-Sepharose column (0.9 X 15 cm) equilibrated in 20 mM Tris-
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