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Mechanistic and metabolic studies of sterol 24,25‐double bond reduction in Manduca sexta
Author(s) -
Short Janel D.,
Guo DeAn,
Svoboda James A.,
Nes W. David
Publication year - 1996
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/(sici)1520-6327(1996)31:1<1::aid-arch1>3.0.co;2-4
Subject(s) - lanosterol , manduca sexta , sterol , desmosterol , biology , biochemistry , sphingidae , campesterol , reductase , microsome , hemolymph , cholesterol , stigmasterol , cholestanol , enzyme , insect , botany , genetics
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 (K m , 3 μM), lanosterol (K m , 18 μM), and cycloartenol (K m , 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‐ 2 H]lanosterol was transformed by larvae to [24‐ 2 H]24,25‐dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and 1 H‐nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25‐reductase activity, 24( R,S ),25‐epimino‐lanosterol (IL), was assayed and found to be inhibitory with an I 50 of 2 μM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (I 50 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‐ 3 H]IL when fed to the larvae. [2‐ 3 H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. © 1996 Wiley‐Liss, Inc.