Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation.
Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation.
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DOI：
10.1016/s0021-9258(18)62872-8
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发表时间：
1970-09
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影响因子：
--
通讯作者：
H. Strobel;A. Y. Lu;J. Heidema;M. J. Coon
中科院分区：
文献类型：
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作者：
H. Strobel;A. Y. Lu;J. Heidema;M. J. Coon
在含血红蛋白P-450的重组肝微粒体酶系统中，脂肪酸、碳氢化合物和药物羟基化所需的热稳定因子已被确定为磷脂酰胆碱。通过在TPNH、氧气、血红蛋白P-450和TPNH-血红蛋白P-450还原酶存在下月桂酸盐、己烷、辛烷、乙基吗啡或苯丙胺羟化的速率来判断，合成的二油酰甘油-3-磷胆碱在取代微粒体因子时是完全活性的。以苯非他明为底物，甘油-3-磷胆碱的各种酰基衍生物在其最佳浓度下的活性依次递增：二硬脂酰；1-单棕榈酰和1-单硬脂酰的混合物；dipalmitoyl;dioleoyl;以及双氯酰和单氯酰的混合物。从厌氧条件下一氧化碳还原差谱确定的TPNH到血红蛋白P-450的电子转移完全依赖于微粒体脂质的存在。停止流动测量表明，在脂质存在时，电子转移速率为双相，快速相基本上在1秒内完成，一阶速率常数约为100 min-1，随后是慢相，一阶速率常数约为6 min-1，但在没有脂质存在时，只能检测到慢相。在类似条件下，但以空气为气相，周转量（每摩尔血红蛋白P-450羟基化苯丙胺的摩尔数）为22分钟-1。这些结果表明，脂质对于酶促血红蛋白P-450的还原是必不可少的，并且只有反应的快速阶段足以支持所观察到的底物羟基化速率。
The heat-stable factor required for fatty acid, hydrocarbon, and drug hydroxylation in a reconstituted liver microsomal enzyme system containing hemoprotein P-450 has been identified as phosphatidylcholine. Synthetically prepared dioleoylglyceryl-3-phosphorylcholine was fully active when substituted for the microsomal factor, as judged by the rate of laurate, hexane, octane, ethylmorphine, or benzphetamine hydroxylation in the presence of TPNH, oxygen, hemoprotein P-450, and TPNH-hemoprotein P-450 reductase. Various acyl derivatives of glyceryl-3-phosphorylcholine showed, at their optimal concentrations, the following increasing order of activity with benzphetamine as the substrate: distearoyl; a mixture of 1-monopalmitoyl and 1-monostearoyl; dipalmitoyl; dioleoyl; and a mixture of dilauroyl and monolauroyl.Electron transfer from TPNH to hemoprotein P-450, as determined from the carbon monoxide reduced difference spectrum under anaerobic conditions, was completely dependent upon the presence of microsomal lipid. Stopped flow measurements showed that the rate of electron transfer was biphasic in the presence of the lipid, with a rapid phase largely completed in less than 1 sec and having a first order rate constant of about 100 min-1, followed by a slow phase having a first order rate constant of about 6 min-1, but only the slow rate could be detected in the absence of the lipid. Under similar conditions, but with air as the gas phase, the turnover number (moles of benzphetamine hydroxylated per mole of hemoprotein P-450) was 22 min-1. These results show that the lipid is essential for the enzymatic reduction of hemoprotein P-450 and that only the rapid phase of the reaction is adequate to support the observed rate of substrate hydroxylation.