Partial Purification and Characterization of Phosphatidic Acid-Specific Phospholipase A(1) in Porcine Platelet Membranes

Biochim Biophys Acta. 2003 Feb 20;1631(1):17-25. doi: 10.1016/s1388-1981(02)00319-0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Platelets / chemistry*
  • Cell Membrane / chemistry
  • Chromatography / methods
  • Electrophoresis, Polyacrylamide Gel
  • Lysophospholipids / biosynthesis
  • Octoxynol
  • Phosphatidic Acids*
  • Phospholipases A / chemistry
  • Phospholipases A / isolation & purification*
  • Phospholipases A / metabolism
  • Substrate Specificity
  • Swine

Substances

  • Lysophospholipids
  • Phosphatidic Acids
  • Octoxynol
  • Phospholipases A