Purification of a novel calcium-independent phospholipase A2 from rabbit kidney.
We have recently identified a cytosolic calcium-independent phospholipase A2 (PLA2) that represents the major measurable PLA2 activity in rabbit proximal tubules (Portilla, D., Shah, S. V., Lehman, P. A., and Creer, M. H.(1994) J. Clin. Invest. 93, 1609-1615). We now report the 3200-fold purification of this PLA2 to homogeneity from rabbit kidney cortex through sequential column chromatography including anion exchange, hydrophobic interaction, Mono Q, hydroxylapatite, phenyl-Sepharose, and chromatofocusing fast protein liquid chromatography from rabbit kidney cortex. The purified enzyme had a molecular mass of 28 kDa, possessed a specific activity of 1.2 micronol/mg min and a neutral pH optimum, and exhibited a preferential hydrolysis toward sn-2 fatty acid from diradylglycerophospholipids. The purified polypeptide hydrolyzed plasmenylcholine > phosphatidylcholine glycerophospholipids and selectively cleaved phospholipids containing arachidonic acid at the sn-2 position in comparison to oleic acid. Antibodies against the purified protein precipitated all of the soluble calcium-independent PLA2 activity from rabbit kidney cortex. These data altogether suggest that the 28-kDa protein in the kidney represents a novel class of calcium-independent PLA2.
Portilla
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Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7199, USA.
Dai
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G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007672:Kidney Cortex / Q000201:enzymology*; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D011817:Rabbits; D013379:Substrate Specificity
D007527:Isoenzymes; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium