Cloning and expression of a human 14-3-3 protein mediating phospholipolysis. Identification of an arachidonoyl-enzyme intermediate during catalysis.
The major phospholipase A2 activity in sheep platelets is mediated by at least three chromatographically resolvable isoforms of a 30-kDa dimeric polypeptide which are responsive to physiologic increments in calcium ion and possess a dramatic substrate selectivity (Loeb, L. A., and Gross, R. W. (1986) J. Biol. Chem. 261, 10467-10470). Herein, we describe the cloning and expression of the human equivalent of one such isoform and demonstrate that it catalyzes the cleavage of the sn-2 fatty acid of choline and ethanolamine glycerophospholipids through the formation of a stable acyl-enzyme intermediate. Transesterification of the sn-2 acyl group of phosphatidylcholine to the recombinant 30-kDa polypeptide is over 50-fold selective for arachidonic acid, is augmented by calcium ion, and results in the formation of an arachidonoyl-thioester intermediate. Homology analysis demonstrated that the polypeptide mediating this transesterification is one member of a family of proteins collectively designated as 14-3-3 proteins. These results demonstrate that at least one intracellular mammalian phospholipase A2 employs a catalytic strategy distinct from that utilized by extracellular phospholipases A2 (i.e. formation of an acyl-enzyme intermediate by nucleophilic attack versus activation of a water molecule) and that arachidonic acid in endogenous phospholipid storage depots can, in principle, be sequentially transferred through an acyl-enzyme intermediate without the prior obligatory release of free arachidonic acid.
Zupan
L A
LA
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Steffens
D L
DL
Berry
C A
CA
Landt
M
M
Gross
R W
RW
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001345:Autoradiography; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008066:Lipolysis; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium