PURIFICATION AND CHARACTERIZATION OF HEMOLYMPH 3-DEHYDROECDYSTEROID 3-BETA-REDUCTASE OF THE SILKWORM, BOMBYX-MORI

3-Dehydroecdysteroid 3 beta-reductase (3-dehydroecdysone 3 beta-reduct ase) is involved in conversion of biologically inactive 3-dehydroecdys one secreted from prothoracic gland to ecdysone, Larval hemolymph of B ombyx mori yielded potent enzyme activity and thus the enzyme was puri fied from Bombyx larval hemolymph by 5320-fold after six step purifica tion. The molecular mass of the enzyme was estimated to be 42,000 Da b ased on SDS-polyacrylamide gel electrophoresis and the optimal pH was 6.6, which was similar to the pH of larval hemolymph, The enzyme requi res NADH or NADPH as cosubstrate and K-m values for NADH and NADPH wer e 5.4 and 0.90 mu M, respectively, showing that NADPH is potent cosubs trate for the hemolymph 3 beta-reductase. Western blot analysis showed that the enzyme was present abundantly in hemolymph, in some measure in hemocytes, fat body, ovary and testis but little or not in other ti ssues examined, which were in accord with the enzyme activity in the e xtracts of those tissues.