肝脏微粒体对香豆素7-羟基化作用的荧光分光光度研究。

A spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes.

作者信息

Creaven P J, Parke D V, Williams R T

出版信息

Biochem J. 1965 Aug;96(2):390-8. doi: 10.1042/bj0960390.

DOI:10.1042/bj0960390

PMID:4378707

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1207052/

Abstract

The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH(2) and oxygen. Activity with NADH(2) was one-third of that with NADPH(2). 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at -12 degrees . Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2'-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.

摘要

已测定了3-羟基香豆素、7-羟基香豆素、7-羟基-4-甲基香豆素和7-甲氧基-4-甲基香豆素的荧光特性。7-羟基香豆素在pH1至9范围内表现出激发态电离。2. 描述了一种灵敏且特异的荧光分析法用于测定7-羟基香豆素（伞形酮），及其在肝匀浆和其他组织制剂中的应用。3. 已用该方法研究了香豆素酶促羟基化生成7-羟基香豆素的过程，并确定了兔肝制剂的最佳条件。酶活性存在于微粒体部分，需要NADPH(2)和氧气。NADH(2)的活性是NADPH(2)的三分之一。4. 添加NADP对于肝脏10000g上清液制剂的充分活性是必要的。制备过程中添加烟酰胺可在-12摄氏度储存的组织中保留辅酶活性。6-磷酸葡萄糖对储存或新鲜组织的活性无影响。5. 对氯汞苯甲酸以及微粒体药物代谢酶的常用抑制剂SKF酸、SKF 525A和礼来7132会产生抑制作用，但2,2'-联吡啶不会。6. 来自兔、豚鼠、河狸鼠、猫和鸽子的肝匀浆表现出活性，但大鼠或小鼠肝脏制剂以及蝗虫脂肪体不会将香豆素羟基化为伞形酮。大鼠肝匀浆和微粒体制剂中不存在该酶系统。此外，大鼠肝脏还含有兔肝香豆素-7-羟化酶系统以及豚鼠肝脏对伞形酮进一步代谢的抑制剂。豚鼠肝脏制剂将香豆素羟基化为伞形酮，然后将该产物转化为其葡糖醛酸苷。7. 雌性兔肝的香豆素-7-羟化酶活性是雄性兔肝的两到三倍。