Purification of a Novel Calcium-independent Phospholipase A2 from Rabbit Kidney*
- From the Division of Nephrology, Department of Medicine, University of Arkansas for Medical Sciences and Veterans Administration Medical Center, Little Rock, Arkansas 72205-7199
- ‡To whom correspondence should be addressed: Dept. of Medicine, Division of Nephrology, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 501, Little Rock, AR 72205-7199. Tel.: 501-660-2030; Fax: 501-671-2503.
Abstract
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 μmol/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.
Footnotes
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↵* This work was supported by National Institutes of Health Grant R29 DK46914 and a Veterans Administration Merit Review Award (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- PLA2
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phospholipase A2
- FPLC
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fast protein liquid chromatography
- PMSF
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phenylmethylsulfonyl fluoride
- PAGE
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polyacrylamide gel electrophoresis
- Bis-Tris
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2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
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- Received January 5, 1996.
- Revision received April 3, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
