Steroidogenic potential of lyophilized mitochondria from bovine adrenocortical tissue.

Abstract

When incubated with [3H]cholesterol, a bovine adrenocortical mitochondrial pellet obtained by centrifugation at 12,000 x g yielded, as expected, only the C2102 metabolites progesterone and pregnenolone. However, the steroidogenic potential of the 12,000 X g pellet fraction was augmented significantly by lyophilization. After 1yophilization, the 12,000 x g pellet converted the sterol into C1, androgens and corticosteroids, in addition to C2102 pregnane derivatives. Leaching the lyophilized mitochondrial fraction with either hexane or acetone increased substantially the yields of the metabolites. It did not change qualitatively the array of metabolites formed during in vitro incubation, but 5a-reductase activity was unmasked by the washings, particularly with acetone. Thus, the fraction sedimented at 12,000 X g contains the complete complement of steroidogenic enzymes required for the biosynthesis of the aforementioned adrenal hormones. These results cast doubt upon the widely held belief that the various enzymes required for adrenocortical steroidogenesis are distributed between two different subcellular compartments, the mitochondrion and the endoplasmic reticulum.have little or no contamination by microsomes. When incubated with [3H]cholesterol as substrate, this pellet catalyzed the formation of only C2102 metabolites from the sterol (see Table 2). But when the pellet was lyophilized and then used for incubation, the Iyophilized pellet was, surprisingly, able to catalyze the conversion of cholesterol to other adrenal steroids as well. The formation ofthese products required the participation of enzymes that were previously not considered to be associated with the mitochondrial fraction. Leaching of the Iyophilized pellet with either hexane or acetone magnified the results, as would be expected if a competing endogenous substrate were removed. Moreover, these leaching processes uncovered the presence in this fraction of 5a-reductase, which also has not previously been thought to be associated with the mitochondrial fraction. Even a pellet obtained by centrifugation at 6500 x g (to further reduce the possibility of microsomal contamination) was, after lyophilization and washing with hexane, able to mimic the activity of the 12,000 x g pellet in its ability to convert [3H]cholesterol to steroids more polar than C2102 steroids.This paper reports that a pellet sedimented at 12,000 x g during centrifugation of a homogenate of bovine adrenocortical tissue has the enzymic capacity to convert in vitro cholesterol into C1902 steroids (androstenedione, dehydroepiandrosterone, and 3f3-hydroxy-5a-androstan-17-one), C21corticosteroids (corticosterone and cortisone), and C2102 metabolites (pregnenolone, progesterone, and 3f3-hydroxy5a-pregnan-20-one). This result would seem to require a reappraisal of the widely accepted belief (1, 2) that the enzymes required for the conversion of the sterol into the adrenal steroids are localized in different subcellular structures, with the side-chain cleavage enzyme, 11f3-hydroxy...

Submitochondrial Distribution of Three Key Steroidogenic Proteins (Steroidogenic Acute Regulatory Protein and Cytochrome P450scc and 3β-Hydroxysteroid Dehydrogenase Isomerase Enzymes) upon Stimulation by Intracellular Calcium in Adrenal Glomerulosa Cells

“…The intramitochondrial presence of 3␤-HSD could add a facilitating step of important functional significance to the Ca 2ϩ -mediated cholesterol transfer: channeling pregnenolone to progesterone should be highly favored by 3␤-HSD located in close proximity to P450 scc and may actually represent a major pathway, in line with the fact that pregnenolone is the preferential substrate of mitochondrial 3␤-HSD (27). In this regard, it is worth mentioning that the existence of multihormonal enzyme complexes ("hormonads") has already been proposed some years ago by Lieberman and Prasad (54) and Prasad et al (55).…”

Effect of Truncated Forms of the Steroidogenic Acute Regulatory Protein on Intramitochondrial Cholesterol Transfer

“…In addition, based on the observations of Papadopoulos and colleagues (42)(43)(44)(45), it is possible that if such a complex exists, it may include the peripheral benzodiazepine receptor and its ligand the diazepam binding inhibitor proteins. Interestingly, the possible existence of a multiprotein complex in steroidogenic mitochondria, referred to as hormonads, was predicted several years ago by Lieberman and colleagues (46,47). It will be of interest to determine whether an association between any or all of these proteins exists in the mitochondrial membrane and, perhaps even more interestingly, whether they exist in contact sites and if this association occurs preferentially with the C-terminal portion of the StAR protein.…”

Other conceivable renditions of some of the oxidative processes used in the biosynthesis of steroid hormones

“…In 1992, Prasad et al reported [14] that when [7-3 H]-cholesterol was incubated with a source of the enzymes present in bovine adrenal mitochondria, they were able to isolate and identify several metabolites, the formation of which can be considered to be in accord with the predicted chemical reactions described in Figs. 5-8.…”