INHIBITION OF PHOSPHOLIPASE-A2 BY CIS-UNSATURATED FATTY-ACIDS - EVIDENCE FOR THE BINDING OF FATTY-ACID TO ENZYME
INHIBITION OF PHOSPHOLIPASE-A2 BY CIS-UNSATURATED FATTY-ACIDS - EVIDENCE FOR THE BINDING OF FATTY-ACID TO ENZYME
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DOI：
10.1016/0005-2760(92)90292-4
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发表时间：
1992-06-22
期刊：
影响因子：
--
通讯作者：
FRANSON, RC
中科院分区：
文献类型：
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作者：
RAGHUPATHI, R;FRANSON, RC
钙依赖性磷脂酶 A2 在体外受到顺式不饱和脂肪酸 (CUFA) 的显着抑制，反式不饱和或饱和脂肪酸的抑制程度要小得多。因此，CUFA 可能充当脂肪分解的内源性抑制剂。为了更好地理解抑制机制，采用动力学分析、荧光光谱和凝胶渗透色谱来证明 CUFA 与来自眼镜蛇毒液的高度纯化的 Ca2+ 依赖性磷脂酶 A2 相互作用。花生四烯酸以明显的竞争性方式抑制[1-C-14]油酸标记的高压灭菌大肠杆菌和[1-C-14]亚油酸标记的磷脂酰乙醇胺的水解。当进行凝胶渗透色谱时，[H-3]花生四烯酸而不是[H-3]棕榈酸与酶共迁移。花生四烯酸和其他 CUFA 使酶的荧光强度以剂量依赖性方式增加了近 2 倍（50-mu-M = 对照的 180%）；花生四烯酸甲酯没有效果。饱和脂肪酸对酶荧光的影响不大（50-mu-M = 对照的 122%）。抑制体外酶活性近 80% 的花生四烯酸浓度不会改变磷脂酶 A2 与大肠杆菌底物的结合。总的来说，这些数据表明，虽然 CUFA 选择性地与酶结合，但它们不会影响磷脂酶 A2-底物相互作用。 CUFA 对体外磷脂酶 A2 活性的抑制可能是通过形成无酶活性的酶-底物-抑制剂复合物来介导的。
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 Ca2+-dependent phospholipase A2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [1-C-14]oleate-labelled, autoclaved Escherichia coli and [1-C-14]linoleate-labelled phosphatidylethanolamine in an apparent competitive manner. When subjected to gel permeation chromatography, [H-3]arachidonate, but not [H-3]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-mu-M = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50-mu-M = 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.