METABOLISM OF MALATE IN BOVINE ADRENOCORTICAL MITOCHONDRIA STUDIED BYC-13-NMR SPECTROSCOPY

C-13-NMR spectroscopy was used to study the metabolism of [C-13]malate in bovine coupled adrenocortical mitochondria. The most apparent diff erence between the mitochondria from steroidogenic tissues and mitocho ndria from other tissues is the presence, in addition to the normal re spiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [C-13]malate was synthesized from [C-13]succin ate by isolated adrenocortical mitochondria. The basic functional susp ension consisted of oxygenated mitochondria to which were added ADP, i norganic phosphate (P-i) and [C-13]malate, both in the absence or pres ence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [C-13]citrate and [C-13]pyruvate from [C-13]malate. The C -13 labeling of these two metabolites demonstrated an important role o f the malic enzyme and the kinetics depended on the presence of the st eroid substrate; the citric acid cycle was stopped during the hydroxyl ation pathway. The addition of cyanide, a strong inhibitor of the resp iratory chain, confirmed an increased malic enzyme activity when hydro xylation occurred, since pyruvate was trapped by formation of a cyanoh ydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the st eroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.