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J. Biol. Chem., Vol. 283, Issue 37, 25428-25436, September 12, 2008

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Identification and Functional Characterization of Adipose-specific Phospholipase A₂ (AdPLA)^*

From the Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720

Phospholipases A₂ (PLA₂s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA₂ that we named AdPLA (adipose-specific phospholipase A₂). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF₃, 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA₂. Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA₂s, group XVI.

Received for publication, May 30, 2008 , and in revised form, July 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant DK075682 (to H. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1.

¹ Both authors contributed equally to this work.

² Recipient of a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

³ To whom correspondence should be addressed: Dept. of Nutritional Science and Toxicology, 219 Morgan Hall, University of California, Berkeley, CA 94720. Tel.: 510-642-3978; Fax: 510-642-0535; E-mail: hsul{at}nature.berkeley.edu.

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