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

我们首先检测了食蟹猴脑微粒体细胞色素P450（P450）的表达水平和催化性质。免疫印迹分析清楚地表明P450在食蟹猴脑微粒体中表达的证据。7-当食蟹猴脑微粒体用作酶源时，也检测到乙氧基香豆素O-脱乙基活性。根据这些结果，我们确定了P450在猴脑中的存在。其次，我们从恒河猴中提取mRNA并制备cDNA文库。尽管使用抗大鼠和人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的，我们尝试从猴肝微粒体中纯化P450。最后从未经处理的食蟹猴肝微粒体中纯化了四种形式的P450（P450 CMLa-d）并进行了表征。从多格拉狒狒， 关于我们 同时，还分离鉴定了与P450 CMLa（P450 3A酶）关系密切的P450 BLa。针对自身P450产生的抗体用于猴脑P450的免疫筛选。但未获得阳性克隆。另一方面，已经确定了编码猴细胞色素P450（P450）2B的cDNA的核苷酸序列。使用从未经处理的食蟹猴的肝微粒体中纯化的抗P450 CMLa的抗体，从未经处理的恒河猴的肝mRNA制备的cDNA文库中分离具有2，275 bp插入片段（Mac 2B）的cDNA克隆。对克隆的插入片段进行测序，发现其含有编码491个氨基酸的多肽的开放阅读框。从推导的氨基酸序列计算的分子量为55，969。由Mac 2B编码的N-末端34个氨基酸与通过纯化的食蟹猴P450 CMLa的Edman降解分析确定的那些相同。在P450 2B亚家族中，Mac 2B的核苷酸和推导的氨基酸序列与人P450 2B 6的核苷酸和推导的氨基酸序列最相似，与人P450 2B 6的核苷酸和推导的氨基酸序列分别为94%和90%。北方印迹分析结果表明，在恒河猴脑组织中检测到与Mac 2B密切相关的mRNA。我们还继续对恒河猴脑中P450 2B的序列进行了分析。少

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