Characterization of monkey cytochrome P450, P450 CMLd, responsible for S-mephenytoin 4-hydroxylation in hepatic microsomes of cynomolgus monkeys.
Characterization of monkey cytochrome P450, P450 CMLd, responsible for S-mephenytoin 4-hydroxylation in hepatic microsomes of cynomolgus monkeys.
复制标题
猴细胞色素 P450、P450 CMLd 的表征，负责食蟹猴肝微粒体中的 S-美芬妥英 4-羟基化。
DOI：
10.1006/abbi.1994.1254
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发表时间：
1994
影响因子：
3.9
通讯作者：
M. Kitada
中科院分区：
文献类型：
--
作者：
S. Ohmori;K. Chiba;H. Nakasa;T. Horie;M. Kitada
我们从食蟹猴肝微粒体中分离到一种新的细胞色素P450（P450），它能够催化S-美芬妥英4 '-羟基化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）判断，最终制备物（称为P450 CMLd）是明显均一的，并且估计该蛋白的最小分子量为53 kDa。P450 CMLd的N-末端氨基酸序列（鉴定为16个残基）与P450 2C 9 cDNA编码的蛋白质的N-末端氨基酸序列完全相同。P450 CMLd与分别从雄性大鼠和人类肝微粒体中纯化的抗P450 2C 11和P450 2C 9的抗体均具有交叉反应性。在食蟹猴的肝微粒体中，两种抗体均识别在SDS-PAGE上显示不同迁移率的两种蛋白质（50和53 kDa）。P450 CMLd是重组体系中S-美芬妥英4 '-羟基化反应的良好催化剂。抗P450 2C 9抗体抑制食蟹猴肝微粒体中S-美芬妥英4 '-羟化酶的活性，但不抑制R-美芬妥英4'-羟化酶以及R-和S-美芬妥英N-脱甲基酶的活性。根据这些证据，我们得出结论，P450 CMLd被归类为P450 2C亚家族，并作为食蟹猴肝微粒体中S-美芬妥英4 '-羟化酶之一。
We isolated a new form of cytochrome P450 (P450) which was able to catalyze S-mephenytoin 4'-hydroxylation from hepatic microsomes of cynomolgus monkeys. The final preparation (referred to as P450 CMLd) was apparently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weight of this protein was 53 kDa. The N-terminal amino acid sequence of P450 CMLd (identified 16 residues) was identical with that of protein encoded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibodies raised against P450 2C11 and P450 2C9 which were purified from hepatic microsomes of male rats and humans, respectively. In hepatic microsomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd was a good catalyst for S-mephenytoin 4'-hydroxylation in a reconstituted system. Anti-P450 2C9 antibody inhibited the activity of S-mephenytoin 4'-hydroxylase, but not the activities of R-mephenytoin 4'-hydroxylase and R- and S-mephenytoin N-demethylases in liver microsomes from cynomolgus monkeys. From these lines of evidence we conclude that P450 CMLd is classified into the P450 2C subfamily and acts as one of the S-mephenytoin 4'-hydroxylases in hepatic microsomes of cynomolgus monkeys.