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Studies on the structure and function of cytochrome P450 in monkey brain

猴脑细胞色素P450结构与功能的研究

基本信息

批准号：
05671807
负责人：
OHMORI Shigeru
金额：
$ 1.34万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671807/
关键词：
Monkey Brain Liver Cytochrome P450 Drug metabolism Microsomes ミクロゾームチトクロームP450

项目摘要

We first examined the expression level of cytochrome P450 (P450) and catalytic properties of brain microsomes of cynomolgus monkeys. Immunobolt analysis clearly indicated the evidence of expression of P450 in brain microsomes of cynomolgus monkeys. 7-Ethoxycoumarin O-deethylation activity was also detected when cynomolgus monkey brain microsomes were used for the enzyme source. From these results, we ascertained the presence of P450 in monkey brain. Next, we extracted mRNA from rhesus monkey and prepared the cDNA library. Although, immunoscreening was performed by using antibodies raised against rat and human P450s, fragment which was encoded the full-length of brain P450 was not obtained. As antigens used for the immunoscreening were not purified from monkey liver microsomes, we tried to purify P450s from hepatic microsomes of monkey. Four forms of P450s were finally purified from hepatic microsomes of untreated cynomolgus monkeys (P450CMLa-d) and characterized. From doguera baboons, … More , P450 BLa which was closely related to P450 CMLa (P450 3A enzyme) was also isolated and characterized. Antibodies raised against the se P450s were used for immunoscreening of monkey brain P450. However, no positive clone was obtained. On the other hand, the nucleotide sequence of a cDNA coding for monkey cytochrome P450(P450)2B has been determined. Using antibody against P450 CMLa which had been purified from hepatic microsomes of untreated cynomolgus monkeys, a cDNA clone with 2,275 bp insert(Mac2B)was isolated from a lambdagt11 cDNA library prepared from hepatic mRNA from an untreated rhesus monkey. The cloned insert was sequenced and found to contain an open reading frame coding for a polypeptide of 491 amino acids. The molecular weight calculated from the deduced amino acid sequence was 55,969. The N-terminal 34 amino acids encoded by Mac2B are identical to those determined by Edman degradation analysis of purified cynomolgus monkey P450 CMLa. The nucleotide and the deduced amino acid sequence of Mac2B were most similar to those of human P450 2B6 among P450 2B subfamily and those sequences of Mac2B were 94% and 90% identical to those of human P450 2B6, respectively. It was also demonstrated that mRNA closely related to Mac2B was detected by Northern blot analysis in rhesus monkey brain. We still continue the sequence analysis for the P450 2B in rhesus monkey brain. Less

我们首先检测了食蟹猴脑微粒体细胞色素P450(P450)的表达水平和催化特性。免疫印迹分析清楚地证明了P450在食蟹猴脑微粒体中的表达。7-乙氧基香豆素O-脱乙基酶以食蟹猴脑微粒体为酶源时也检测到了活性。根据这些结果，我们确定了P450在猴脑中的存在。接下来，我们从恒河猴中提取了mRNA，并制备了文库。虽然利用针对大鼠和人P450的抗体进行了免疫筛选，但没有获得编码脑P450全长的片段。由于用于免疫筛选的抗原不是从猴肝微粒体中纯化出来的，因此我们尝试从猴肝微粒体中纯化P450。最终从未经处理的食蟹猴(P450CMLa-d)肝微粒体中纯化出四种形式的P450，并对其进行了鉴定。来自Doguera巴布亚，…此外，还分离并鉴定了与P450CMLa(P4503A酶)密切相关的P450BLA。用抗Se P450的抗体对猴脑P450进行免疫筛选。但未获得阳性克隆。另一方面，测定了猴细胞色素P450(P450)2B编码基因的核苷酸序列。利用从未经处理的食蟹猴肝微粒体中提纯的抗P450 CMLa的抗体，从未经处理的恒河猴肝组织中制备的lambdagt11基因文库中，获得了一个含有2,275bp插入片段(Mac2B)的cDNA克隆。对克隆的插入片段进行了测序，发现该插入片段含有一个编码491个氨基酸的多肽的开放阅读框。根据推导的氨基酸序列计算出其相对分子质量为55,969。Mac2B编码的N端34个氨基酸与纯化的食蟹猴P450CMLa的Edman降解分析结果一致。在P450 2B亚家族中，Mac2B的核苷酸和氨基酸序列与人P450 2B6最为相似，与人P450 2B6的同源性分别为94%和90%。Northern印迹分析也证实了与Mac2B密切相关的基因在恒河猴脑中的表达。我们仍在继续对恒河猴脑中P450 2B的序列分析。较少