McNamara B C, Jefcoate C R
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Arch Biochem Biophys. 1989 Nov 15;275(1):53-62. doi: 10.1016/0003-9861(89)90349-4.
Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.
体内预先用促肾上腺皮质激素（ACTH）刺激可增强离体大鼠肾上腺线粒体中的胆固醇侧链裂解（CSCC）（8倍）。通过将来自ACTH刺激的肾上腺的胞质溶胶添加到未刺激大鼠的线粒体中，这种刺激的一部分在体外得以保留（2.5至6倍）。体内用环己酰亚胺（CX）处理可完全抑制体内反应，并将体外胞质溶胶刺激分解为几个组分：（i）ACTH敏感、CX敏感；（ii）ACTH敏感、CX不敏感；以及（iii）ACTH不敏感、CX不敏感。这些组分对ACTH胞质溶胶刺激的贡献大致相等。组分（i）和（iii）很可能分别对应于先前鉴定的胞质成分类固醇生成激活肽和固醇载体蛋白2（SCP2）。通过放射免疫测定或刺激7-脱氢胆固醇还原酶的能力测定，ACTH处理后肾上腺胞质溶胶或其他亚细胞组分中的SCP2并未升高。用抗SCP2 IgG处理从胞质溶胶中完全去除SCP2，可使胞质溶胶刺激活性增加，增加量与ACTH或CX处理无关。添加相当于胞质溶胶中存在量的SCP2可使SCP2耗尽的胞质溶胶恢复活性，但单独添加或与完整胞质溶胶一起添加时均无作用，表明胞质溶胶中存在一种增强SCP2作用的因子。纯肝SCP2刺激CX线粒体CSCC 1.5至2倍（EC50为0.7 microM），但效力仅为肾上腺胞质溶胶中SCP2的五分之一。在这些制剂中区分出了两部分反应性胆固醇，分别以琥珀酸支持的活性和额外的异柠檬酸支持的活性为特征。ACTH胞质溶胶和SCP2各自刺激胆固醇向线粒体P450scc的一部分的可利用性，该部分可被琥珀酸还原，但未能刺激向仅由异柠檬酸还原的额外P450scc的可利用性。
