STRUCTURE AND REGULATION OF CYTOCHROMES P-450

我们建议纯化肾上腺皮质微粒体细胞色素P-450 （17 α-羟化酶; 21-羟化酶）。 这些酶将 表征为蛋白质（纯度，MW，等电点），血红素 蛋白质（光谱，底物诱导的差异光谱，血红素含量）和 作为酶（动力学、抑制剂）。 将注意比较 肾上腺17 α-羟化酶与猪睾丸微粒体P-450， 催化17-Alapha-羟基化和C（17，20）-裂解酶，并且我们有 最近净化。 我们计划纯化猪细胞色素P-450 来自小球藻和束状藻的11 β-/18-羟化酶 因为我们之前的研究表明， 不同的酶。 胆固醇侧链断裂 酶（以前从牛肉中纯化）也将从猪中纯化 肾上腺皮质线粒体 这些酶将依次测序， 自动化微测序和克隆（共同主要研究者J.E. 夏夫利和B。分别为沃利斯）。 与此同时， 这些酶将通过放射性亲和烷基化来标记， 天然类固醇底物的溴乙酰氧基衍生物，以及 烷基化的氨基酸将被识别和定位在主要的 通过序列分析进行结构分析。 异烯醇酮结合蛋白 微粒体和线粒体内膜中的胆固醇 在目前的支持期内。 这两种蛋白质将 现在被分离和鉴定。 三种蛋白质的有限水解 微粒体细胞色素P-450（睾丸羟化酶/裂解酶;肾上腺 21-羟化酶和肾上腺17 α-羟化酶）将用于分离 使用P-450的结合片段在P-450上的还原酶结合域 黄素蛋白还原酶附着在琼脂糖珠上。 纯化的 将比较不同P-450的还原酶结合结构域， 这些肽在相关蛋白质的一级结构中的位置 第450章一定要确定 该提案的协作扩展将 包括核磁共振和晶体学的详细研究。

We propose to purify adrenocortical microsomal cytochromes P-450 (17Alpha-hydroxylase; 21-hydroxylase) from pig. The enzymes will be characterized as proteins (purity, MW, isoelectric points), as heme proteins (spectra, substrate-induced difference spectra, heme content) and as enzymes (kinetics, inhibitors). Attention will be paid to comapring adrenal 17Alpha-hydroxylase with pig testicular microsomal P-450, which catalyzes both 17Alapha-hydroxylation and C(17,20)-lyase, and which we have recently purified. We plan to purify porcine cytochrome P-450 11Beta-/18-hydroxylase from the zona glomerulosa and zona fasiculata separately because our previous studies suggest that theree are two different enzymes in the cortex. The cholesterol side-chain cleavage enzyme (previously purified from beef) will also be purified from pig adrenocortical mitochondria. These enzymes will be sequenced in turn using automated microsequencing and cloning (Co-Principal Investigators J.E. Shively and B. Wallis, respectively). At the same time, active sites of these enzymes will be labeled by radioactive affinity alkylation using bromoacetoxy derivatives of the natural steroid substrates, and the alkylated amino acids will be identified and localized in the primary structure by the sequence studies. Binding proteins for pregnenolone in microsomes and for cholesterol in the inner mitochondrial membrane have been identified in the present period of support. These two proteins will now be isolated and characterized. Limited proteolysis of the three microsomal cytochromes P-450 (testicular hydroxylase/lyase; adrenal 21-hydroxylase and adrenal 17Alpha-hydroxylase) will be used to isolate a reductase-binding domain on P-450 using the binding fragment of the P-450 flavoprotein reductase attached to sepharose beads. The purified reductase-binding domains of different P-450's will be compared and the location of these peptides in the primary structures of the relevant P-450's will be determined. Collaborative extensions of the proposal will involve detailed studies by NMR and crystallography.