Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (Km, 3 μM), lanosterol (Km, 18 μM), and cycloartenol (Km, 33 μM) to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4S-tritium labelled NADPH. [24-2H]lanosterol was transformed by larvae to [24-2H]24,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and 1H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,S),25-epiminolanosterol (IL), was assayed and found to be inhibitory with an I50 of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I50, 60 ppm). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-3H]IL when fed to the larvae. [2-3H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques.
|Number of pages||22|
|Journal||Archives of Insect Biochemistry and Physiology|
|State||Published - 1996|
- Manduca sexta
- Tobacco hornworm