Mitochondrial Transhydrogenations in Manduca sexta: Relationship between Reversible NADPH → NAD+ Transhydrogenase and Ecdysone 20-Monooxygenase in Fifth Instar Larvae
Date of Award
Doctor of Philosophy (Ph.D.)
Carmen Fioravanti, PhD
Raymond Larsen, PhD (Committee Member)
Martin Mitchell, PhD (Committee Member)
Mark Munson, PhD (Committee Member)
Jill Zeilstra-Ryalls, PhD (Committee Chair)
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 non energy- and energy-linked activity. Biochemical characterization for reversibility, energy-linkages, pH optima, stability to dialysis/heat denaturation, transmembrane proton translocation and localization were accomplished. During the ten day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the 50-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations are apparent. Using isolated mitochondrial membranes, the M. sexta transhydrogenase was subjected to kinetic analysis pertaining to the NADPH → NAD+ as well as non energy-linked and the ATP-dependent, energy linked NADH → NADP+ reactions. Kinetic analysis demonstrated that the reversible insect transhydrogenase is subject to site-specific inhibition, contains two substrate binding sites (viz., NADP(H) and NAD(H)), and is susceptible to end-product inhibition. The effect of various allelochemicals on the M. sexta transhydrogenations was also evaluated. Taken together, the findings of this dissertation support a distinct physiological role of mitochondrial transhydrogenase in M. sexta post-embryonic development.
Vandock, Kurt, "Mitochondrial Transhydrogenations in Manduca sexta: Relationship between Reversible NADPH → NAD+ Transhydrogenase and Ecdysone 20-Monooxygenase in Fifth Instar Larvae" (2010). Biology Ph.D. Dissertations. 36.