Co-Authors:
Weirich, G.F., Insect Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, United States
Thompson, M.J., Insect Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, United States
Svoboda, J.A., Insect Hormone Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, United States
Abstract:
Five enzyme activities in midgut cytosol of Manduca sexta last instar larvae are potentially involved in the interconversion of 3β-hydroxyecdysteroids, 3-oxoecdysteroids, and 3α-hydroxyecdysteroids. A Sephadex G-25-filtered high-speed supernatant was used to determine some of the characteristics of the corresponding enzymes. The pH optima of ecdysone oxidase and NADH-dependent 3-oxoecdysteroid 3α-reductase were 7.5, the pH of the midgut cytosol was 7.9. The apparent kinetic parameters for the NADH-dependent 3α-reductase were Km (for NADH) = 80.8 ± 10.8 μM and Vmax = 0.58 ± 0.30 nmol/min/mg protein, and for the NADPH-dependent 3-oxoecdysteroid 3β-reductase, Km (for NADPH) = 19.3 ± 2.5 μM and Vmax = 4.39 ± 0.40 nmol/min/mg protein. NAD+ and NADP+ inhibited the enzymatic 3-oxoecdysteroid reductions, but the reactions were not reversible (i.e. no conversion of ecdysone or 3-epiecdysone to 3-dehydroecdysone). Sodium chloride (0.2 M) inhibited the 3α-reductase activity with NADH and strongly increased the 3α-reductase activity with NADPH. © 1991.