ENZYMES OF ECDYSTEROID 3-EPIMERIZATION IN MIDGUT CYTOSOL OF MANDUCA SEXTA: pH OPTIMA COSUBSTRATE KINETICS, AND SODIUM CHLORIDE EFFECT*

-Five enzyme activities in midgut cytosol of Manduca sexta last instar larvae are potentially involved in the interconversion of 3fl-hydroxyecdysteroids, 3-oxoeedysteroids, and 3ct-hydroxyeedysteroids. A Sephadex G-25-filtered high-speed supernatant was used to determine some of the characteristics of the corresponding enzymes. The pH optima of eedysone oxidase and NADH-dependent 3-oxoeedysteroid 3u-reductase were 7.5, the pH of the midgut cytosol was 7.9. The apparent kinetic parameters for the NADH-dependent 3~t-reductase were Km (for NADH)=80.8+ 10.8#M and Vm~x = 0.58 +__ 0.30 nmol/min/mg protein, and for the NADPH-dependent 3-oxoecdysteroid 3fl-reductase, K m (for NADPH) = 19.3 ___ 2.5 gM and V~ = 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 3~-reductase activity with NADH and strongly increased the 3~t-reductase activity with NADPH. Key Word Index: Ecdysone oxidase; 3-oxoeedysteroid 3-reductase; 3oepiecdysone; 3-dehydroeedysone; 3a-hydroxyecdysteroid; 3fl-hydroxyeedysteroid; Manduca sexta; midgut; cytosol; NADH; NADPH; sodium chloride