Mitochondrial transhydrogenations of Manduca sexta: developmental and biochemical characterization

Midgut mitochondria from wandering stage fifth larval instar Manduca sexta have been shown to exhibit a transhydrogenase that catalyzes the following reversible reaction:

NADPH + NAD⁺ ↔ NADP⁺ + NADH.

The NADPH-forming transhydrogenation (i.e the NADH→NADP⁺ reaction) occurs as a nonenergy- and energy-linked activity, with energy for the latter derived from the electron transport-dependent oxidation of NADH (ETD) or succinate (SD), or by ATP hydrolysis via Mg⁺⁺-dependent ATPase (ATPD). The mitochondrial transhydrogenase of M. sexta midgut tissue is membrane-associated, optimal at pH 7.5, stable to prolonged dialysis, thermally labile, and a transmembrane proton translocator. During postembryonic development in M. sexta, the conversion of the molting hormone, ecdysone, to its physiologically active form, 20-hydroxyecdysone (20-HE), is essential. The formation of 20-HE is catalyzed by a cytochrome P450 dependent steroid hydroxylase, ecdysone 20-monooxygenase (E20-M). Mitochondrial E20-M preferentially uses NADPH as the reductant and this NADPH could arise from the reversible mitochondrial transhydrogenase found in M. sexta midgut tissue. The present study centers on the evaluation of transhydrogenations during the developmental period preceding the larval-pupal molt (fifth larval instar) of M. sexta. The study revealed peaks of transhydrogenase activity on day five coinciding with the peaks in E20-M during the same period. Significant increases in M. sexta transhydrogentations on day five were noted in both midgut and fatbody tissues. This data suggests an interesting correlation between E20-M, NADPH utilization and the need for reducing equivalents as supplied by a reversible, energy-linked NADPH-forming transhydrogenase.