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Analyzes of Structure and Function of Novel Carbonyl Metabolic Enzymes : MALDO and MALCO

新型羰基代谢酶 MALDO 和 MALCO 的结构和功能分析

基本信息

批准号：
06672208
负责人：
YAMAMOTO Ikuo
金额：
$ 1.34万
依托单位：
Hokuriku 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：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06672208/
关键词：
Aldehyde Alcohol Oxygenase Cannabinoid Cloning MALDO MALCO P450 アルコーナ

项目摘要

Three P450 isozymes, named P450MDX-A,P450GPF-A and P450JM-C,which are major isozymes responsible for 9-anthraldehyde microsomal aldehyde oxygenase (MALDO) activity were purified from hepatic microsomes of mice, guinea pigs and Japanese monkeys. The N-terminal amino acid sequences of these isozymes suggest that the isozymes are member of CYP2B subfamily. Piperonal, alpha-n-amylcinnamaldehyde, vanillin and tolaldehyde were also oxidized to the corresponding carboxylic acids by MALDO of mouse liver.Three P450 isozymes, named P450MDX-B,P450GPF-B and P450JM-E,which are major isozymes responsible for 7-OH-DELTA^8-tetrahydrocannabinol microsomal alcohol oxygenase (MALCO) activity were purified from hepatic microsomes of mice, guinea pigs and female Japanese monkeys. The N-terminal amino acid sequences of these isozymes suggest that the isozymes are member of CYP3A subfamily.A cDNA encoding a P450 isozyme was isolated from a cDNA library constructed from male 129 mouse liver. The cDNA,designated Cyp2c-29 (129), may correspond to a polymorphic variant of Cyp2c-29 (isolated from C57BL/6 mouse liver cDNA library). One out of the six nucleotide changes is nonconservative, resulting in the replacement of 106 Ile of Cyp2c-29 (C57BL/6) by 104 Met. The comparison with Cyp2c-29 sequence shows 4 differences concentrated within a 174-nucleotide sequence located 228-405. mRNAs corresponding to this region (188-445) in C57BL/6, ddN,ddY,C3H,DBA/2 and ICR mouse livers were amplified by the methods of RT-PCR.The amplified nucleotide sequences were the identical with those of Cyp2c-29 (129) except for C57BL/6. A cDNA clone encoding P450MDX-B was isolated from a mouse liver cDNA library. The nucleotide sequence of the cDNA is identical to that of Cyp3a-11.

从小鼠、豚鼠和日本猴的肝微粒体中纯化出三种P450同工酶，分别为P450MDX-A、P450GPF-A和P450JM-C，它们是负责9-蒽醛微粒体醛加氧酶(MALDO)活性的主要同工酶。这些同工酶的 N 端氨基酸序列表明这些同工酶是 CYP2B 亚家族的成员。胡椒醛、α-正戊基肉桂醛、香草醛和甲苯醛也被小鼠肝脏的MALDO氧化成相应的羧酸。三种P450同工酶，命名为P450MDX-B、P450GPF-B和P450JM-E，它们是负责7-OH-DELTA^8-四氢大麻酚的主要同工酶从小鼠、豚鼠和雌性日本猴的肝微粒体中纯化出微粒体醇加氧酶（MALCO）活性。这些同工酶的N-末端氨基酸序列表明这些同工酶是CYP3A亚家族的成员。从雄性129小鼠肝脏构建的cDNA文库中分离出编码P450同工酶的cDNA。该 cDNA 称为 Cyp2c-29 (129)，可能对应于 Cyp2c-29 的多态性变体（从 C57BL/6 小鼠肝脏 cDNA 文库中分离）。六个核苷酸变化中有一个是非保守的，导致 Cyp2c-29 (C57BL/6) 的 106 个 Ile 被 104 个 Met 取代。与 Cyp2c-29 序列的比较显示，4 个差异集中在位于 228-405 的 174 个核苷酸序列内。采用RT-PCR方法在C57BL/6、ddN、ddY、C3H、DBA/2和ICR小鼠肝脏中扩增该区域(188-445)的mRNA。扩增的核苷酸序列除C57BL/6外与Cyp2c-29(129)的核苷酸序列相同。从小鼠肝脏 cDNA 文库中分离出编码 P450MDX-B 的 cDNA 克隆。该cDNA的核苷酸序列与Cyp3a-11的核苷酸序列相同。