The Stability of Mitochondrial d-β-Hydroxybutyric Dehydrogenase and Its Relationship to the Respiratory Chain

Abstract

Earlier work in this laboratory (1) has shown that the diphosphopyridine nucleotide-linked n-P-hydroxybutyric dehydrogenase of animal tissues (2) is localized exclusively in the mitochondria. The enzyme is very firmly attached to the mitochondrial membranes (cf. l-5), in contrast to other pyridinelinked dehydrogenases of mitochondria, which are usually readily extracted after disruption of structure by means of sonic oscillations, chemical treatment, or hypotonicity. There is evidence that mitochondrial n-fi-hydroxybutyric dehydrogenase bears a special relationship to the components of the respiratory chain. Gamble and Lehninger (6) found that the bound diphosphopyridine nucleotide in digitonin fragments of rat liver mitochondrial membranes (7) is reactive with bound n-P-hydroxybutyric dehydrogenase, but it does not interact with other dehydrogenases present; the bound diphosphopyridine nucleotide is also unreactive with added alcohol dehydrogenase. Oxidation of o-P-hydroxybutyrate via bound DPN occurs with coupled formation of adenosinet.riphosphate; on the other hand, interaction of /3-hydroxybutyric dehydrogenase with added or “external” DPN produces nonphosphorylating electron transport (8). Devlin and Bedell (9) have shown that mitochondrial /3-hydroxybutyric dehydrogenase may, under certain conditions, serve as a “shuttle” system for the introduction of reducing equivalents from extramitochondrial DPNH to the phosphorylating respiratory chain. For these and other reasons, it was suggested that n-fl-hydroxybutyric dehydrogenase may serve some unique function in the respiratory chain and that the bound DPN with which it reacts is specifically concerned in phosphorylating electron transport (1, 10). In this paper, further evidence for a specific relationship between n-P-hydroxybutyric dehydrogenase and the respiratory chain is reported; this evidence was obtained from examination of the rather peculiar stability characteristics of the enzyme. n-/3-Hydroxybutyric dehydrogenase as it occurs in either sonic or digitonin fragments of rat liver mitochondria is relatively stable anaerobically, but is reversibly inactivated under aerobic conditions. The enzyme is almost completely protected from aerobic inactivation by the combined addition of succinate, DPN+, and ATP. When the dehydrogenase is inactivated aerobically, it can be reactivated again by incubation with thiols; however, the reactivation requires addition of oxidized DPN and reductants * Supported by grants from the National Institutes of Health, the National Science Foundation, the Nutrition Foundation, and the Whitehall Foundation. t Henry Strong Denison Scholar, 1959-1961. of the respiratory chain. These and other findings indicate a functional relationship between an essential thiol group or groups of P-hydroxybutyric dehydrogenase, the binding of DPN, and the action of succinate on the respiratory chain.