Regulation of Sex-Specific Forms of Cytochrome P-450 : Cytochrome P-450 in Liver Microsomes of Hamsters

细胞色素 P-450 性别特异性形式的调节 : 仓鼠肝微粒体中的细胞色素 P-450

• 批准号: 63490001
• 负责人: KAMATAKI Tetsuya
• 金额: $ 3.39万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63490001/
• 关键词: Cytochrome P-450; Hamster; Liver microsomes; Drug Metabolizing enzyme; Sex difference; Purification; チトクロ-ムP-450; ハムスター; 肝ミクロゾーム; チトクロームP-450

Three forms of cytochrome P-450 were purified to electrophoretical homogeneity on SDS-PAGE. One of the forms of cytochrome P-450 (P-450-fHS1) purified from liver microsomes of female hamster was considered to be mainly present in a low spin state as judged by absolute spectrum of the oxidized form and was cross-reactive with anti-P450-male antibodies. Other forms of cytochrome P-450 Purified from liver microsomes of male and female hamsters according to the cross-reactivity with anti-P-450-male antibodies were tentatively termed P-450-mHS2 and P-450-fHS2, respectively. Chromatographic behavior of P-450-mHS2 on DEAE-5PW column and hydroxylapatite column were identical to those of P-450-fHS2. Judging from the absolute spectra, both cytochromes P-450 were shown to be mainly in low spin states. N-Terminal amino acid sequence of P-450-mHS2 was identical with that of P-450-fHS2 when the first 20 amino acid sequence was compared. In addition, N-terminal amino acid sequence of P-450-fHS2 was f … More ound to be 80% homologous to those of P-450-mHS2 and P-450-fHS2. Western blot analysis of liver microsomes of male hamsters with anti-P-450-fHS1 antibodies showed that P-450-fHS1 or cytochrome P-450 immunochemically related to P-450-fHS1 was also present in liver microsomes of male hamsters. However, the amounts of P-450-fHS1 was found to be much higher in female than male. On the contrary, the content of cytochrome P-450 cross-reactive with anti-P-450-mHS2 antibodies in female hamster microsomes was comparable to that in male hamster microsomes. The content of P-450-fHS1 in liver microsomes of female hamsters was decreased by gonadectomy of female hamsters or the administration of testosterone. Treatment of gonadectomized female hamsters with testosterone, but not with estradiol, resulted in further decrease in the amount of P-450-fHS1. On the other hand, the content of P-450-fHS1 or cytochrome P-450 immunochemically related to P-450-fHS1 in male hamster microsomes was virtually unaffected by gonadectomy or the treatment with estradiol whereas the content of the cytochrome P-450 was increased by the treatment of gonadectomized male hamsters with estradiol. Both P-450-fHS2 and P-450-fHS2 were active for 6beta-hydroxylation of progesterone but not for testosterone hydroxylation. P-450-fHS2 catalyzed N-demethylations of aminopyrine, benzphetamine and erythromycin, and O-deethylation of 7-ethoxycoumarin in a recontituted system. P-450-mHS2 catalyzed N-demethylation of erythromycin and O-deethylation 7-ethoxycoumarin but not N-demethylations of aminopyrine and benzphetamine. Less

三种形式的细胞色素P-450在SDS-PAGE上纯化到电泳均匀性。从雌性仓鼠肝微粒体中纯化的细胞色素P-450 （P-450- fhs1）的一种形式，从氧化形式的绝对光谱判断，认为主要以低自旋状态存在，并与抗p450 -雄性抗体发生交叉反应。其他形式的细胞色素P-450根据与抗P-450雄性抗体的交叉反应性从雄性和雌性仓鼠肝微粒体中纯化，暂定命名为P-450- mhs2和P-450- fhs2。P-450-mHS2在DEAE-5PW柱和羟基磷灰石柱上的色谱行为与P-450-fHS2相同。从绝对光谱来看，两种细胞色素P-450主要处于低自旋态。P-450-mHS2的n端氨基酸序列与P-450-fHS2的前20个氨基酸序列完全一致。此外，P-450-fHS2的n端氨基酸序列与P-450-mHS2和P-450-fHS2同源性达80%以上。用抗P-450- fhs1抗体对雄性仓鼠肝微粒体进行Western blot分析，发现在雄性仓鼠肝微粒体中也存在P-450- fhs1或与P-450- fhs1免疫化学相关的细胞色素P-450。然而，P-450-fHS1在女性中的含量比男性高得多。相反，与抗P-450- mhs2抗体交叉反应的细胞色素P-450在雌性仓鼠微粒体中的含量与雄性仓鼠微粒体中的含量相当。雌性仓鼠肝微粒体中P-450-fHS1的含量通过雌性仓鼠性腺切除或睾酮处理而降低。用睾酮而不是雌二醇治疗去性腺的雌性仓鼠，导致P-450-fHS1的数量进一步下降。另一方面，雄性仓鼠微粒体中P-450- fhs1或与P-450- fhs1免疫化学相关的细胞色素P-450的含量几乎不受性腺切除或雌二醇处理的影响，而细胞色素P-450的含量在性腺切除的雄性仓鼠中被雌二醇处理后增加。P-450-fHS2和P-450-fHS2对孕酮的6 -羟基化有活性，但对睾酮的羟基化无活性。P-450-fHS2在重组体系中催化氨基比林、苯丙胺和红霉素的n -去甲基化和7-乙氧基香豆素的o -去甲基化。P-450-mHS2能催化红霉素的n -去甲基化和7-乙氧基香豆素的o -去甲基化，但不能催化氨基吡啶和苯丙胺的n -去甲基化。少

项目成果

Masayuki Komori,Kanako Nishio,Hiroaki Ohi,Mitsukasu Kitada and Tetsuya Kamataki: "Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome Pー450." J.Biochem.105. 161-163 (1989)

Masayuki Komori、Kanako Nishio、Hiroaki Ohi、Mitsukasu Kitada 和 Tetsuya Kamataki：“含有人胎儿肝细胞色素 P-450 完整编码区的 cDNA 的分子克隆和序列分析，J.Biochem.161-163”。

Masayuki Komori, Kanako Nishio, Hiroaki Ohi, Mitsukazu Kitada and Tetsuya Kamataki: "Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome P-450." J. Biochem.105. 161-163 (1989)

Masayuki Komori、Kanako Nishio、Hiroaki Ohi、Mitsukazu Kitada 和 Tetsuya Kamataki：“含有人胎儿肝细胞色素 P-450 完整编码区的 cDNA 的分子克隆和序列分析。”

Hiroaki Ohi, Miyuki Iwasaki, Masayuki Komori, Toshiaki Miura, Mitsukazu Kitada, Shoryo Hayashi and Testuya Kamataki: "Effects of serum testosterone level with buserelin on the activities of drug and testosterone hydroxylases and on the content of a male-s

Hiroaki Ohi、Miyuki Iwasaki、Masayuki Komori、Toshiaki Miura、Mitsukazu Kitada、Shoryo Hayashi 和 Testuya Kamataki：“布舍瑞林对血清睾酮水平对药物和睾酮羟化酶活性以及男性睾酮含量的影响

Kazuhide Ohta,Michiyo Motoya,Masayuki Komori,Toshiaki Miura,Mitsukazu Kitada and Tetsuya Kamataki: "Interspecies homology of cylochrome P-450:Purification and toxicological significance of a high spin form of cytochrome P-450(P-450-D2)from liver microsome

Kazuhide Ohta、Michiyo Motoya、Masayuki Komori、Toshiaki Miura、Mitsukazu Kitada 和 Tetsuya Kamataki：“细胞色素 P-450 的种间同源性：来自肝脏的细胞色素 P-450 (P-450-D2) 高自旋形式的纯化和毒理学意义

Kazuhide Ohta, Michiko Motaya, Masayuki Komori, Toshiaki Miura, Mitsukazu Kitada and Tetsuya Kamataki: "Interspecies homology of cytochroem P-450 : Purification and toxicological significance of a high spin form of cytochrome P-450 (P-450-D2) from liver m

Kazuhide Ohta、Michiko Motaya、Masayuki Komori、Toshiaki Miura、Mitsukazu Kitada 和 Tetsuya Kamataki：“细胞色素 P-450 的种间同源性：来自肝脏的细胞色素 P-450 (P-450-D2) 高自旋形式的纯化和毒理学意义