Solubilization and properties of Ca2+-dependent human platelet phospholipase A2.
Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.
D000418:Albumins / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D002848:Chromatography, DEAE-Cellulose; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000097:blood*; D054467:Phospholipases A2; D010742:Phospholipid Ethers; D013379:Substrate Specificity
D000418:Albumins; D002413:Cations, Divalent; D005960:Glucosides; D010713:Phosphatidylcholines; D010742:Phospholipid Ethers; C018619:octyl-beta-D-glucoside; C043051:1-O-hexadecyl-2-arachidonyl-sn-glycero-3-phosphocholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium