Grace K J, Zavortink D, Jacobs R S
Department of Biological Sciences, University of California, Santa Barbara 93106.
Biochem Pharmacol. 1994 Apr 20;47(8):1427-34. doi: 10.1016/0006-2952(94)90343-3.
A sesquiterpene furanoic acid (SFA) marine natural product isolated from soft corals of the genus Sinularia (Bowden et al., Aust J Chem 36: 371-376, 1983) was found to inactivate bee venom phospholipase A2 (bvPLA2, EC 3.1.1.4) in vitro. In this study, we characterized the kinetics of inactivation of bvPLA2 by this compound. The apparent IC50 value was 0.5 microM, and the inactivation of bvPLA2 was time dependent. The drug-enzyme binding appeared to be of a non-competitive, high-affinity nature that was irreversible by aqueous dialysis. The inactivation was prevented by the simultaneous addition of excess lysophosphatidylcholine (lysoPC) during the initial binding step, suggesting that modification of the enzyme by SFA occurs at or near the substrate binding site. Activation of bvPLA2 was observed with lysoPC addition at concentrations equimolar to bvPLA2 and higher. Saturation of activation occurred at concentrations greater than 10 microM lysoPC, and preincubation of bvPLA2 with 100 microM lysoPC did not inhibit the enzyme. Analysis of the post-incubation mixture of SFA-inhibited enzyme in the presence of lysoPC revealed the presence of unaltered enzyme exhibiting typical Michaelis-Menten kinetics. The significance of these observations is discussed in light of the recent discussion by Ortiz on the manoalide binding site on bvPLA2.
从Sinularia属软珊瑚中分离出的一种倍半萜呋喃酸（SFA）海洋天然产物（Bowden等人，《澳大利亚化学杂志》36: 371 - 376, 1983）在体外被发现可使蜂毒磷脂酶A2（bvPLA2，EC 3.1.1.4）失活。在本研究中，我们对该化合物使bvPLA2失活的动力学进行了表征。表观IC50值为0.5微摩尔，bvPLA2的失活具有时间依赖性。药物 - 酶结合似乎是非竞争性、高亲和力性质的，通过水透析不可逆。在初始结合步骤中同时添加过量溶血磷脂酰胆碱（lysoPC）可防止失活，这表明SFA对酶的修饰发生在底物结合位点或其附近。在添加等摩尔及更高浓度的lysoPC时观察到bvPLA2的激活。当lysoPC浓度大于10微摩尔时激活达到饱和，并且将bvPLA2与100微摩尔lysoPC预孵育不会抑制该酶。在lysoPC存在下对SFA抑制的酶的孵育后混合物进行分析，发现存在表现出典型米氏动力学的未改变的酶。根据Ortiz最近关于bvPLA2上的 manoalide结合位点的讨论，对这些观察结果的意义进行了讨论。
