Isolation of a human myocardial cytosolic phospholipase A2 isoform: Fast atom bombardment mass spectroscopic and reverse-phase high pressure liquid chromatography identification of choline and ethanolamine glycerophospholipid substrates

Stanley L. Hazen, Curtis R. Hall, David A. Ford, Richard W. Gross

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Abstract

Recent studies have demonstrated the existence of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in canine myocardium that have been implicated as enzymic mediators of ischemic membrane damage. We now report that human myocardium contains two functionally distinct isoforms of cytosolic calcium-independent phospholipase A2. The major cytosolic phospholipase A2 isoform preferentially hydrolyzes plasmalogen substrate, possesses a pH optimum of 7.0, and is chromatographically resolvable from a minor cytosolic calcium-independent phospholipase A2 isoform that hydrolyzes plasmenylcholine and phosphatidylcholine substrates at similar rates and possesses a pH optimum of 8.5. The major cytosolic calcium-independent phospholipase A2 isoform was identified as a 40-kD polypeptide after its 182,000-fold purification by sequential column chromatographies to a final specific activity of 67 μmol/mg · min. The purified 40-kD human myocardial phospholipase A2 preferentially hydrolyzes plasmalogens containing arachidonic acid at the sn-2 position. Both reverse-phase HPLC and fast atom bombardment mass spectroscopic analysis of human myocardial ethanolamine and choline glycerophospholipids demonstrated that plasmenylethanolamine and plasmenylcholine molecular species containing arachidonic acid at the sn-2 position are prominent constituents of human myocardium. Collectively, these results identify and characterize the major human myocardial cytosolic calcium-independent phospholipase A2 activity, demonstrate the presence of functionally distinct human myocardial cytosolic calcium-independent phospholipase A2 isoforms, and document the abundance of arachidonoylated plasmalogen molecular species in human myocardium that serve as substrates.

Original languageEnglish
Pages (from-to)2513-2522
Number of pages10
JournalJournal of Clinical Investigation
Volume91
Issue number6
DOIs
StatePublished - Jun 1993

Keywords

  • Arachidonic acid
  • Myocardial ischemia
  • Phospholipase A
  • Phospholipids
  • Plasmalogens

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