Inhibition of phospholipase A2 by cis-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme.
Calcium-dependent phospholipases A2 are markedly inhibited in vitro by cis-unsaturated fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca(2+)-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-14C]oleate-labelled, autoclaved Escherichia coli and [1-14C]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonate, but not [3H]palmitate, comigrated with the enzyme. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 microM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 microM = 122% of control). Concentrations of arachidonate that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A2 to the E. coli substrate. Collectively, these data demonstrate that, while CUFAs selectively bind to the enzyme, they do not influence phospholipase A2-substrate interaction. Inhibition of in vitro phospholipase A2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-substrate-inhibitor complex.
Raghupathi
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R
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614.
Franson
R C
RC
D016718:Arachidonic Acid / Q000378:metabolism; D002850:Chromatography, Gel; D004546:Elapid Venoms / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D004546:Elapid Venoms; D005231:Fatty Acids, Unsaturated; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2