Partial purification and characterization of phosphatidic acid-specific phospholipase A(1) in porcine platelet membranes.
We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A(1) type (PA-PLA(1)) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA(1) was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca(2+) and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA(1) in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA(2) inhibitor, a Ca(2+)-independent phospholipase A(2) inhibitor, and a DG lipase inhibitor.
Miyazawa
Daisuke
D
Department of Preventive Nutraceutical Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuhoku, Nagoya, 467-8603, Japan.
Ikemoto
Atsushi
A
Fujii
Yoichi
Y
Okuyama
Harumi
H
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D008246:Lysophospholipids / Q000096:biosynthesis; D017830:Octoxynol; D010712:Phosphatidic Acids; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D008246:Lysophospholipids; D010712:Phosphatidic Acids; D017830:Octoxynol; D010741:Phospholipases A