Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from ACTH-treated rats: differential responses to different reducing precursors.
Heterogeneous pools of cholesterol side-chain cleavage activity in adrenal mitochondria from ACTH-treated rats: differential responses to different reducing precursors.
复制标题
ACTH 治疗大鼠肾上腺线粒体中胆固醇侧链裂解活性的异质池：对不同还原前体的不同反应。
DOI：
10.1016/0303-7207(90)90125-r
复制
发表时间：
1990
影响因子：
4.1
通讯作者：
Jefcoate,CR
中科院分区：
文献类型：
--
作者：
McNamara,BC;Jefcoate,CR
从 ACTH 治疗的大鼠中分离出的肾上腺线粒体中内源性胆固醇的侧链裂解 (SCC) 表明反应性胆固醇库的大小取决于还原性前体。在还原剂的最佳浓度下，异柠檬酸盐的池量通常是琥珀酸盐的至少 2 倍。琥珀酸盐支持的反应快速完成，对 2 分钟预孵育高度敏感，并且未能耗尽光谱检测到的 P-450scc-胆固醇复合物。含有 1 mM 异柠檬酸盐的胆固醇 SCC 表现出快相代谢增加 2-3 倍、明显的慢相、对预孵育不敏感以及光谱检测到的胆固醇-P-450scc 复合物消耗 60%。无论是在均质化过程中还是直接在孵育过程中添加牛血清白蛋白 (BSA) 和 EDTA，都可以防止琥珀酸响应造成的预孵育损失，并消除琥珀酸和异柠檬酸活性之间的大部分差异。 BSA/EDTA 的这种作用可在 5 分钟内被辛酸逆转，其机制可通过 Ca2+ 增强。这些独特的还原剂特征表明，只有线粒体亚群或单个线粒体内的活性池可以用琥珀酸盐支持胆固醇 SCC，而异柠檬酸盐对于其余部分是必需的。琥珀酸支持的代谢对预孵育或辛酸的快速反应表明胆固醇代谢的关键因子被耗尽。添加 20α-羟基胆固醇或脱氧皮质酮的代谢表明，在琥珀酸支持的胆固醇代谢停止或预孵育后，NADPH 仍然完全可用。因此，孕烯醇酮形成的停止是由于胆固醇供应失败，而不是 NADPH 不足。预孵育效应表明能量依赖性成分的损失，该成分可增强胆固醇的供应。测试的一种可能性是 GTP，一种膜间胆固醇转移的激活剂（Xu et al. (1989) J. Biol. Chem. 264, 17674–17680）正在丢失。添加 GTP 会稍微激活琥珀酸支持的孕烯醇酮生产，但不能防止预孵育引起的损失。 α-酮戊二酸可生成基质 GTP，是一种有效的还原剂，与琥珀酸结合可防止预孵育引起的损失。
Side-chain cleavage (SCC) of endogenous cholesterol in adrenal mitochondria isolated from ACTH-treated rats indicates that the size of the reactive cholesterol pool depends on the reducing precursor. At optimal concentrations of reductant, this pool was typically at least 2 times greater for isocitrate than for succinate. Succinate-supported reactions were rapidly completed, were highly sensitive to a 2-min preincubation, and failed to deplete spectrally detectedP-450scc-cholesterol complexes. Cholesterol SCC with 1 mM isocitrate exhibited 2–3 times more fast-phase metabolism, a pronounced slow phase, insensitivity to preincubation, and 60% depletion of spectrally detected cholesterol-P-450scccomplexes. Addition of bovine serum albumin (BSA) and EDTA, either during homogenization or directly to the incubation, prevented preincubation losses in response to succinate and removed most of the difference between succinate and isocitrate activities. This effect of BSA/EDTA was reversed within 5 min by octanoate by a mechanism that was enhanced by Ca2+. These distinct reductant characteristics suggest that only a subpopulation of mitochondria or of pools of activity within individual mitochondria can support cholesterol SCC with succinate while isocitrate is necessary for the remainder. The rapid responses of succinate-supported metabolism to preincubation or to octanoate suggest depletion of a critical factor for cholesterol metabolism. Metabolism of added 20α-hydroxycholesterol or deoxycorticosterone established that NADPH remained fully available after succinate-supported cholesterol metabolism had stopped or after preincubation. Cessation of pregnenolone formation, therefore, results from a failure to supply cholesterol, not inadequate NADPH. The preincubation effect suggests loss of an energy-dependent component that enhances this supply of cholesterol. One possibility tested was that GTP, an activator of intermembrane cholesterol transfer (Xu et al. (1989) J. Biol. Chem. 264, 17674–17680), was being lost. Added GTP slightly activated succinate-supported pregnenolone production but did not prevent preincubation-induced losses. α-Ketoglutarate, which can generate matrix GTP, is an effective reductant that, in combination with succinate, prevents preincubation-induced losses.