Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5-bisphosphate
Kinetic analysis in mixed micelles of partially purified rat brain phospholipase D activity and its activation by phosphatidylinositol 4,5-bisphosphate
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DOI：
10.1023/a:1022422418388
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发表时间：
1998-05-01
影响因子：
4.4
通讯作者：
Liscovitch, M
中科院分区：
文献类型：
--
作者：
Chalifa-Caspi, V;Eli, Y;Liscovitch, M
用1-棕榈酰-2-[6-N-（2-甲氧基苯基）-2-甲基-N-（2-甲氧基苯基）-1-甲基-N-（2-甲氧基苯基）-2-（2-甲氧基苯基）-（7-硝基苯并-2-氧杂-1，3-二唑-4-基）氨基]己酰基-磷脂酰胆碱（NBD-PC）和Triton X-100，在Triton X-100对PLD活性具有表面稀释效应且催化速率取决于表面浓度的条件下（以摩尔比表示）。磷脂酰肌醇4，5-二磷酸（PIP 2）特异性激活PLD活性，激活与PIP 2摩尔比的曲线符合Michaelis-Menten方程，K-act值在摩尔比0.001-0.002之间。在0.01的PIP 2摩尔比下观察到最大活化。当部分纯化的PLD以及膜结合的PLD的活性被确定为对纯NBD-PC胶束时，得到类似的值。在混合胶束体系中，PIP 2显示出使PLD对NBD-PC的特异性常数（K-A，其与V-max/K-m成比例）提高6-22倍。动力学分析的PLD转磷脂酰化活性对乙醇，1-丙醇和I-丁醇揭示了米氏-Menten型依赖于酒精浓度高达1000，200和80 mM，分别。虽然V-max值对所有三种醇是相似的，酶的亲和力增加的醇是较长的，和K-m值为乙醇，1-丙醇和1-丁醇是291，75和16 mM（分别）。PLD对乙醇、异丙醇和正丁醇的专一性常数（K-A）分别是对竞争底物水的260、940和5,920倍。l-丙醇和l-丁醇分别在400和100 mM以上抑制PLD活性。目前的结果表明，部分纯化的PLD遵守表面稀释动力学方面，其磷脂底物PC和它的辅因子PIP 2，并在醇的存在下，其transphosphatidylation活性可以分析作为一个竞争性反应的水解反应。
A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of 1-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP2), and the curve of activation versus PIP2 molar ratio fitted a Michaelis-Menten equation with a K-act, value between molar ratios of 0.001-0.002. Maximal activation was observed at a PIP2 molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP2 was shown to elevate by 6-22 fold the specificity constant of PLD towards NBD-PC (K-A, which is proportional to V-max/K-m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, l-propanol and I-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V-max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K-m values for ethanol, l-propanol and I-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K-A) towards ethanol, I-propanol and l-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. l-Propanol and l-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP2, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.