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 (17α-羟基酶；21-羟基酶)。这些酶将会是 以蛋白质(纯度、相对分子质量、等电点)、血红素为特征 蛋白质(光谱、底物诱导差异光谱、血红素含量)和 作为酶(动力学、抑制剂)。我们将注意比较 肾上腺17α-羟基酶与猪睾丸微粒体P-450的关系 催化17Alapha-羟化和C(17，20)裂解酶，我们有 最近提纯的。我们计划纯化猪细胞色素P-450 球状带和束状带的11β-/18-羟基酶 分开是因为我们之前的研究表明有两个 大脑皮层中的不同酶。胆固醇侧链断裂 酶(之前从牛肉中提纯)也将从猪中提纯 肾上腺皮质线粒体。这些酶将依次进行测序，使用 自动微测序和克隆(联合首席调查员J.E. 分别是Shively和B.Wallis)。与此同时，活跃的站点 这些酶将通过放射性亲和烷基化反应进行标记 天然类固醇底物的溴乙酰氧基衍生物，以及 烷基化氨基酸将被鉴定和定位在初级 结构通过对序列的研究。孕烯醇酮结合蛋白 线粒体内膜上的微粒体和胆固醇 在本支助期内已确定。这两种蛋白质将 现在是孤立的和有特点的。三者的有限蛋白分解 微体细胞色素P-450(睾丸羟基酶/裂解酶；肾上腺 21-羟基酶和肾上腺17-羟基酶)将用于分离 利用P-450的结合片段研究P-450上的还原酶结合域 附着在琼脂糖珠上的黄素蛋白还原酶。纯净的 将比较不同的P-450‘S的还原酶结合结构域，并对 这些多肽在相关的一级结构中的位置 P-450‘S将被确定。该提案的协作性扩展将 包括核磁共振和结晶学的详细研究。

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.