草鱼肝微粒体的提取及CYP酶活性的测定

Cytochrome P450 contents and activity in hepatic microsome of grass carp(Ctenopharyngodon idellus)

CYP酶代谢是药物生物转化的主要途径,其数量和活性大小直接影响药物在体内的活化与代谢。我们对草鱼肝微粒体CYP酶含量及其活性进行了初步研究,以差速离心法提取草鱼肝微粒体,以CO还原差示光谱法测得CYP酶及细胞色素b5含量分别为0.619±0.102 nmol/mg、0.264±0.042 nmol/mg。以7-乙氧异吩噁唑酮-O-脱乙基反应、苯胺-4-羟化反应、氨基比林-N-脱甲基反应作为CYP1A、CYP2E、CYP3A的探针反应,测得EROD酶活为0.043±0.004 nmol/mg/min,ANH酶活为0.028±0.002 nmol/mg/min,AMND酶活为0.207±0.035 nmol/mg/m in。结果表明草鱼肝微粒体中CYP酶发育完好,并且具有参与药物代谢的3种主要亚型活性,其含量与活性大小与其它实验动物相差较大。本实验的方法与结果为草鱼CYP酶的系统研究提供可靠手段,最终为指导水产合理用药提供理论依据。

Cytochrome P450（CYP） is one of the most important representatives of the drugs metabolism, and play a key role in the metabolic activation of numerous drugs. This article dealed with the primary study on hepatic microsomal CYP in grass carp. Hepatic microsome was prepared by different ultracentrifugation, the contents of CYP and cytochrome b5 were determined 0. 619 ±0. 102 nmol/mg, 0. 264 ±0. 042 nmol/mg. To use probe substrates 7-ethoxyresorufin-O-deethylation, 4-Aniline-hydroxylation, aminopyrine-N-demethylaion specific for CYP1A, CYP2E, CYP3A, the activity of EROD, ANH, AMND were determined 0.043 ± 0. 004 nmol/mg/min, 0. 028 ± 0. 002 nmol/mg/min, 0. 207 ± 0. 035 nmol/mg/min, respectively. The results showed that three of the main CYP reaction were found in grass carp liver microsomes, the contents of CYP and enzymatic activity were different as compared with the results of other animals reported formerly. The present experimental method and result will facilitate detection of more cytochrome P450 enzyme activities in grass carp, and will be available to administer the medicine properly for aquatic animal.