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.
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 mumol/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.
Hazen
S L
SL
Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Hall
C R
CR
Ford
D A
DA
Gross
R W
RW
D016718:Arachidonic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000032:analysis; D010714:Phosphatidylethanolamines / Q000032:analysis; D010741:Phospholipases A / Q000302:isolation & purification*; D054467:Phospholipases A2; D010955:Plasmalogens / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity
D007527:Isoenzymes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010955:Plasmalogens; C032459:choline plasmalogens; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2