Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA).
Phospholipases A(2) (PLA(2)s) 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 PLA(2) that we named AdPLA (adipose-specific phospholipase A(2)). 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, AACOCF(3), 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 PLA(2). 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 PLA(2)s, group XVI.
Duncan
Robin E
RE
Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720.
Sarkadi-Nagy
Eszter
E
Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720.
Jaworski
Kathy
K
Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720.
Ahmadian
Maryam
M
Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720.
Sul
Hei Sook
HS
Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720. Electronic address: hsul@nature.berkeley.edu.
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology*; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D019556:COS Cells; D002522:Chlorocebus aethiops; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D008246:Lysophospholipids / Q000737:chemistry; D051379:Mice; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry; D054467:Phospholipases A2 / Q000737:chemistry* / Q000378:metabolism; D013440:Sulfides / Q000494:pharmacology
D000577:Amides; D001095:Arachidonic Acids; D005231:Fatty Acids, Unsaturated; D008246:Lysophospholipids; D010713:Phosphatidylcholines; D013440:Sulfides; C081565:arachidonyltrifluoromethane; C059778:7,7-dimethyl-5,8-eicosadienoic acid; D054467:Phospholipases A2; C532349:adipose-specific phospholipase A2, rat