Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase).
Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase).
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来自新生猪睾丸的微粒体细胞色素 P-450。
DOI：
10.1016/s0021-9258(19)69538-4
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
P. Hall
中科院分区：
文献类型：
--
作者：
S. Nakajin;P. Hall
经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和兔抗兔抗血清琼脂双扩散法鉴定，新生猪睾丸微粒体中的细胞色素P-450纯化为均一组分。该酶具有17α-羟基酶和C17，20裂解酶的活性(Vmax=4.6nmol/min/nmol的P-450，Km=1.5微米)和C17，20裂解酶(Vmax=2.6nmol/min/nmol的P-450的产物，Km=2.4微米)。这两种活性都需要NADPH和黄素蛋白P-450还原酶；这些研究使用的是猪和大鼠肝脏的微粒体P-450还原酶。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和十二烷基硫酸钠-Sephadex柱层析法测定了该酶的单亚基分子量为59,000+/-1,000。该酶是一种糖蛋白，含有8nmol的血红素/毫克蛋白质和40nmol的磷脂/毫克蛋白质。通过对还原的P-450一氧化碳络合物的差示光谱分析，所有用吡啶血色素原检测到的血红素都被解释为P-450。该络合物在448 nm处有最大吸收，没有P-420的存在。这些研究提出了一种可能性，即一种微粒体蛋白(细胞色素P-450)可能具有两种酶活性(羟基酶和裂解酶)。
A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).