Membrane topology of guinea pig cytochrome P450 17α revealed by a combination of chemical modifications and mass spectrometry
Membrane topology of guinea pig cytochrome P450 17α revealed by a combination of chemical modifications and mass spectrometry
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DOI：
10.1021/bi035096z
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发表时间：
2003-12-16
期刊：
影响因子：
2.9
通讯作者：
Kominami, S
中科院分区：
文献类型：
--
作者：
Izumi, S;Kaneko, H;Kominami, S
内质网膜上的细胞色素p450参与了外源性和内源性疏水底物的羟基化作用。这些反应需要nadph -细胞色素P450还原酶在同一细胞膜上提供电子。细胞膜在细胞色素P450的反应中起重要作用。研究了豚鼠P450 17alpha的膜拓扑结构，研究了它们在洗涤剂溶解状态和蛋白脂质体对亲水性化学修饰试剂的反应性差异。从大肠杆菌中纯化重组豚鼠细胞色素P450 17α，并将其纳入脂质体膜中。在pH为9.0的条件下，用乙酸酐将溶解在洗涤剂中的赖氨酸残基和蛋白脂质体中的赖氨酸残基乙酰化，在pH为5.0的条件下，用偶联剂1-乙基-3-（3-二甲氨基丙基）盐酸碳二亚胺将酸性氨基酸残基与甘氨酸酰胺偶联。改性是在不诱导P420变性的条件下进行的。修饰后的P450 17alpha's经胰蛋白酶酶切，用MALDI-TOF质谱法测定肽片段的分子量。根据肽分子量的增加，可以推断出修饰的位置。在洗涤剂溶解状态下，11个赖氨酸残基和7个酸性氨基酸残基被修饰，其中位于29、59、490和492位的赖氨酸残基和位于211、212和/或216位的酸性残基未被修饰。n端结构域和c端结构域以及假定的F-G环都位于P450 17alpha的膜结合结构域内或附近。
Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N-and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.