ABSTRACT The mitochondrial, inner-membrane–associated, reversible NADPH → NAD + transhydrogenase of the energetically anaerobic adult cestode Hymenolepis diminuta connects NADPH generation, via a mitochondrial NADP + -specific “malic” enzyme, with NADH formation needed for electron transport. In reducing the pyridine nucleotide, the enzyme concomitantly catalyzes transmembrane proton translocation, thereby coupling NADH formation to ATP generation or NADPH formation to ATP hydrolysis. Detergent-solubilized transhydrogenase, from isolated mitochondrial membranes, was purified to apparent homogeneity using ion exchange and hydroxylapatite chromatographies. The enzyme displayed a monomeric M r of ∼110 kDa and required phospholipid, without which activity was rapidly lost. Of the phospholipids examined, phosphatidylcholine was the most effective. Transhydrogenase-catalyzed NADH formation was inhibited by NAD(P) + and adenylates, suggesting regulatory effects of the pyridine nucleotides and effects of pyridine nucleotide-simulating molecules. In keeping with its proton-translocating function, the enzyme was inhibited by dicyclohexylcarbodiimide. The isolated enzyme catalyzed neither NADH → NADP + nor NADH → NAD + transhydrogenations, thereby suggesting a need for a minimal coupling to electron transport for the NADH → NADP + reaction as well as enzyme specificity. Anti-transhydrogenase monospecific antibodies proved inhibitory to NADPH → NAD + transhydrogenation catalyzed by both isolated and membrane-associated enzymes. This purification study apparently represents a first for parasitic helminths or multicellular invertebrates generally and establishes a framework for evaluating the transhydrogenase as a potential site for specific chemotherapeutic attack.

Although lacking an NADPH→NAD + transhydrogenase system, the essentially energetically anaerobic mitochondria of the adult intestinal nematode Ascaris suum display an inner membrane-associated NADH→NAD + transhydrogenation reaction. This reaction is considered to be reflective of a mechanism(s) that acts in catalyzing a transmembrane translocation of reducing equivalents from NADH in the intermembrane space to matrix NAD + , thereby forming matrix NADH that would serve in electron transport. Ascarid mitochondrial lipoamide dehydrogenase rather than an NADH→NAD + transhydrogenase system has been viewed as the predominant source of inner membrane-associated NADH→NAD + transhydrogenation activity. However, the present study made apparent yet another source of mitochondrial, inner membrane–associated NADH→NAD + activity in A. suum , viz., NADH dehydrogenase. This was made evident via comparisons of the A. suum mitochondrial NADH→NAD + transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities in terms of pH effects, thermal labilities, the involvement of NADH dehydrogenase in the activities of mitochondrial, membrane-associated rotenone-insensitive and rotenone-sensitive NADH-dependent cytochrome c reductases, and mitochondrial membrane versus mitochondrial soluble localizations. Studies of the responses of the NADH→NAD + transhydrogenation, rotenone-insensitive and rotenone-sensitive cytochrome c reductases, and lipoamide dehydrogenase activities to inhibition by copper and cadmium lent additional support to the catalysis of an NADH→NAD + transhydrogenation activity by NADH dehydrogenase. Collectively, the data presented are consistent with an additional physiological catalysis of an NADH→NAD + transhydrogenation in A. suum mitochondria by an inner membrane NADH dehydrogenase component of the rotenone-sensitive cytochrome c reductase system, i.e., the NADH dehydrogenase component of the electron transport system. Comparisons of the A. suum data with those from other essentially anaerobic helminth parasites as well as free-living eukaryotic mitochondrial systems are noted.