MECHANISTIC AND METABOLIC STUDIES OF STEROL 24,25-DOUBLE BOND REDUCTION IN MANDUCA-SEXTA

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-r eductase. From the midgut of fifth instar larvae fed a mixture of sito sterol and campesterol a microsome-bound 24,25-sterol reductase was pr epared that transformed desmosterol (K-m, 3 mu M), lanosterol (K-m, 18 mu M), and cycloartenol (K-m, 33 mu M) to cholesterol, 24,25-dihydrol anosterol, and cycloartanol, respectively. With desmosterol as substra te, the microsome-bound enzyme was found to incorporate tritium into c holesterol from 4S-tritium labelled NADPH. [24-H-2]lanosterol was tran sformed by larvae to [24-H-2]24,25-dihydrolanosterol (structure confir med by mass spectroscopy (MS) and H-1-nuclear magnetic resonance spect roscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epiminolanosterol (IL), was assayed and found to be inhibit ory with an I-50 of 2 mu M. IL was supplemented in the diet of M. sext a with either sitosterol or stigmasterol and found to inhibit developm ent ( (I-50, 60 ppm). The major sterol which accumulated in the Ii-tre ated larvae was desmosterol, confirming the site of inhibition was red uction of the 24,25-bond. IL was converted to [2-H-3]IL when fed to th e larvae. [2-H-3]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. (C) 1996 Wiley-Liss, Inc.