Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta

Abstract

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.

Effects of 20-Hydroxyecdysone on the Reversible Mitochondrial Transhydrogenase in the Tobacco Hornworm,Manduca sexta

“…As reported in Vandock et al (2008), isolated midgut mitochondria from M. sexta catalyzed an NADPH-NAD + transhydro-genation as measured by AcPyAD reduction (Table 1). This NADH-forming reaction was the most active transhydrogenation observed.…”

“…Spectrophotometric assessments of transhydrogenase were performed essentially as described by Fioravanti et al (1992) and Vandock et al (2008). Activities were measured by following acetylpyridine-NAD(P) (AcPyAD(P)) reduction at 375 nm.…”

“…In the tobacco hornworm, Manduca sexta (L.) (Lepidoptera: Sphingidae), the mitochondrial transhydrogenase has been characterized as a membrane-associated, phospholipid-dependent, and energy-linked enzyme that contributes to NADPH synthesis and larval development (Vandock et al 2011). With regard to M sexta , the mitochondrial transhydrogenase has been shown to facilitate the reversible reaction noted here (Vandock et al 2008):…”

Sequence and Structure of the Tobacco Hornworm, Manduca sexta, Transhydrogenase Gene

“…[8][9][10] Indeed, careful experimentation has shown that fatbody and midgut tissues display increased levels Agarose gel of pCr products of M. sexta transhydrogenase fragment using degenerate primers. notes: Degenereate primers (5′ TAYCAYCANGTNTGGGGN3′ and 5′ rAACATrTCNArCATNCK3) designed against the conserved polypeptide sequences YhTVWG and rMLDMFK (Fig.…”

“…9,10 The developmental significance of these transhydrogenations and the role(s) they may play in postembryonic development was evaluated. 8 This transhydrogenase appears to be physiologically linked to the developmentally necessary NADPH-requiring mitochondrial E20-M.…”