METABOLISM IN-VITRO OF CHOLESTEROL AND 25-HYDROXYCHOLESTEROL BY THE LARVAL PROTHORACIC GLANDS OF MANDUCA-SEXTA

The prothoracic glands in vitro convert 25-hydroxycholesterol (25C) to 25-hydroxy-7-dehydrocholesterol (7d25C) and to ecdysteroids at a grea ter rate than cholesterol (C) is converted to ecdysteroids via 7-dehyd rocholesterol (7dC). Mediated via a cytochrome P-450 most probably loc ated in the endoplasmic reticulum (ER), both intact and extensively ho mogenized prothoracic glands, as well as crude subcellular fractions, were able to 7,8-dehydrogenate 25C to 7d25C eight-fold more efficientl y than they could convert C to 7dC. However, less than a twofold diffe rence was observed in the subsequent monooxygenase mediated conversion of these two intermediates formed irt situ into ecdysteroids, mainly ecdysone (E) and 2-deoxyecdysone (2dE) and/or their 3-dehydroderivativ es. When 7dC, and particularly 7d25C, were made directly available to these tissue preparations, their conversion to ecdysteroids greatly ex ceeded that of the ill situ conversion of either C or 25C, via 7dC or 7d25C, respectively. Indeed, there was an eight-fold increase in the V -max for 25C dehydrogenation by homogenized glands relative to the deh ydrogenation of C. PI lost important, however, was the 1000-fold incre ase in the V-max observed for the direct production of E from emulsifi ed 7d25C by gland homogenates relative to E production from 25C via 7d 25C synthesized in situ. Thus, it is apparent that even after the rapi d and efficient conversion of 25C to 7d25C within the ER, the subseque nt rate of conversion of this intermediate to E is greatly retarded re lative to that observed following the direct incubation of emulsified 7d25C with gland homogenates. These differential kinetics of direct an d indirect 7d25C incorporation into E are interpreted as evidence for the existence of a barrier to the efficient translocation of the Delta (5,7)-sterol intermediates from the ER to another site where the subse quent, uncharacterized initial conversions leading to ecdysteroids tak e place. On the basis of studies on mammalian adrenal cortical steroid ogenesis, this site is postulated to be the inner membrane/matrix of t he mitochondria; The present data support the hypothesis that the tran slocation of both 7dC and 7d25C, first from the site of their probable synthesis within the ER membranes, next through the cytosol to the ou ter mitochondrial membrane, and then across the intramitochondrial aqu eous space to the inner membrane/matrix compartment, may be analogous to the translocation in the adrenal cortex of ER-derived C, first to t he plasma membrane and/or to the outer mitochondrial membrane and then to the inner mitochondrial membrane/matrix for P-450scc-mediated conv ersion into pregnenolone. Copyright (C) 1996 Elsevier Science Ltd