Perrin A, Gout E, Chambaz E M, Defaye G
Institut National de la Santé et de la Rechereche Médicale, Départment de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Eur J Biochem. 1994 Jul 1;223(1):51-9. doi: 10.1111/j.1432-1033.1994.tb18965.x.
13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.
采用¹³C-核磁共振光谱法研究了[¹³C]苹果酸在牛肾上腺皮质偶联线粒体中的代谢情况。类固醇生成组织的线粒体与其他组织的线粒体之间最明显的差异在于，除了正常的呼吸链外，还存在负责类固醇羟基化的第二条电子传递系统。[¹³C]苹果酸由分离的肾上腺皮质线粒体从[¹³C]琥珀酸合成。基本功能悬浮液由充氧的线粒体组成，在不存在或存在类固醇底物脱氧皮质酮的情况下，向其中添加二磷酸腺苷（ADP）、无机磷酸盐（Pi）和[¹³C]苹果酸。这些线粒体从[¹³C]苹果酸合成了[¹³C]柠檬酸和[¹³C]丙酮酸。这两种代谢物的¹³C标记表明苹果酸酶具有重要作用，且动力学取决于类固醇底物的存在；在羟基化途径中柠檬酸循环停止。添加呼吸链的强抑制剂氰化物后，由于丙酮酸通过形成氰醇而被捕获，证实了羟基化发生时苹果酸酶活性增加。通过在基本悬浮液中补充未标记的外源代谢物，如丙酮酸或草酰乙酸，比较了在不存在或存在类固醇底物的情况下苹果酸酶和异柠檬酸脱氢酶的相对酶活性。
