Williams-Smith D L, Simpson E R, Barlow S M, Marrison P J
Biochim Biophys Acta. 1976 Oct 13;449(1):72-83. doi: 10.1016/0005-2728(76)90008-6.
Low and high spin ferric cytochrome P-450 and reduced adrenal ferredoxin (adrenodoxin) have been directly studied by EPR techniques in whole rat adrenal glands. The spectra obtained correspond closely to those obtained from sub-cellular fractions except in the case of low spin ferric cytochrome P-450, where there are differences in the shape of the g = 2.41 line. The relative magnitudes of these peaks in anaerobic and aerobic rapidly frozen adrenals from control and corticotropin stimulated hypophysectomised rats were used to investigate the control and rate limiting steps in adrenal steroid biosynthesis via cytochrome P-450. All adrenals showed a close to maximal level of reduced adrenodoxin and aerobic and anaerobic glands from control rats and aerobic glands from corticotropin stimulated rats showed similar quantities of low spin ferric cytochrome P-450. On anaerobiosis the quantity of low spin ferric cytochrome in adrenals from corticotropin stimulated rats dropped to 30--40% of the aerobic level. Treatment of the rats with cycloheximide prior to administration of corticotropin prevented these changes. Approximately 0.4% of the total cytochrome P-450 was high spin ferric in control adrenals and in aerobic stimulated adrenals this rose to approximately to 0.6%. These results demonstrate that association of substrate with cytochrome P-450 is the rate limiting step in adrenal steroidogenesis via cytochrome P-450. It is suggested on the basis of these and mitochondrial optical and EPR experiments that the limiting step being observed is cholesterol binding to cholesterol side chain cleavage cytochrome P-450, and that the rate of this association is stimulated by corticotropin.
利用电子顺磁共振（EPR）技术，对完整大鼠肾上腺中的低自旋和高自旋铁细胞色素P-450以及还原型肾上腺铁氧化还原蛋白（肾上腺皮质铁氧还蛋白）进行了直接研究。除了低自旋铁细胞色素P-450的情况外，所获得的光谱与从亚细胞组分获得的光谱非常接近，在低自旋铁细胞色素P-450的情况下，g = 2.41线的形状存在差异。来自对照和促肾上腺皮质激素刺激的垂体切除大鼠的厌氧和好氧快速冷冻肾上腺中这些峰的相对大小，被用于研究通过细胞色素P-450的肾上腺类固醇生物合成中的控制和限速步骤。所有肾上腺均显示出接近最大水平的还原型肾上腺皮质铁氧还蛋白，对照大鼠的需氧和厌氧腺体以及促肾上腺皮质激素刺激大鼠的需氧腺体显示出相似数量的低自旋铁细胞色素P-450。在厌氧条件下，促肾上腺皮质激素刺激大鼠肾上腺中的低自旋铁细胞色素数量降至需氧水平的30 - 40%。在给予促肾上腺皮质激素之前用环己酰亚胺处理大鼠可防止这些变化。对照肾上腺和需氧刺激肾上腺中总细胞色素P-450的约0.4%为高自旋铁，在需氧刺激肾上腺中这一比例升至约0.6%。这些结果表明，底物与细胞色素P-450的结合是通过细胞色素P-450的肾上腺类固醇生成中的限速步骤。基于这些以及线粒体光学和EPR实验表明，所观察到的限速步骤是胆固醇与胆固醇侧链裂解细胞色素P-450的结合，并且这种结合的速率受到促肾上腺皮质激素的刺激。
