Effects ofin Vivoandin VitroAdministered Thyroxine on Substrate Metabolism of Isolated Rabbit Ventricle Mitochondria. III. Substrate Effects on Pyridine Nucleotide Reduction, on the Reversal of Electron Transport, and on the “Respiratory Control by ATP”1
Ventricle mitochondria have been isolated and assayed from normal and from thyroxine-treated, initially normal rabbits (20 μg T4 administered sc per 100 g body wt per day, for 1, 4, or 14 consecutive days). Repeated T4 treatment in vivo generally increased the net rate and degree of “succinate-linked” NAD reduction, as it did the QO2 of controlled and active states in the presence of succinate. After 4–14 T4 injections in vivo, the “succinate-linked” controlled state showed neither ATP requirement for NAD reduction nor respiratory inhibition by ATP, whereas both these phenomena were observed prior to hormone treatment. Nevertheless, in the succinate-linked active state, ATP inhibited the QO2 and augmented the NAD reduction, unaffected by T4 treatment. Rotenone inhibited the “succinate-linked” NAD reduction in all preparations. D( + ) Malate inhibited the “succinatelinked” NAD reduction irrespective of T4 treatment. Ninety second preincubation of euthyroid mitochondria with cysteine sulfinate, pyruvate, or octanoate yielded “thyrotoxic” controlled state kinetics upon succinate addition: high QO2, marked NAD reduction without ATP added, and no respiratory control by ATP. Oxaloacetate addition to the preincubation prevented these effects. Moreover, oxaloacetate addition to thyrotoxic mitochondria (4–;14 T4 injections) reestablished the “euthyroid” kinetics: decreased the “succinate-linked” QO2, and restored both NAD reduction and respiratory control by ATP in the controlled state. By contrast, neither bovine serum albumin, thyroxine-binding prealbumin, Rutamycin, nor dinitrophenol, trifluoro- CCP, nor T4 yielded such kinetic transformation in vitro. Unaffected by in vivo T4 administration, ATP yielded NAD reduction and strongest respiratory control with added substrates such as L( _ ) malate + malonate or pyruvate + malate; succinate addition or malonate omission decreased the QO2 control by ATP. In contrast to other substrates (malate, succinate), the NAD reduction “linked to” pyruvate disappeared within 24 hr after T4 injection. The data are consistent with tight coupling of oxidative phosphorylation in all mitochondria here examined (see preceding paper). It is proposed that in these ventricle mitochondria the citric acid cycle substrates (malate-oxaloacetate) strongly affected the respiratory control by ATP. (Endocrinology89:385, 1971)