The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.

Abstract

After pregnenolone is produced in mitochondria, conversion to glucocorticoid and mineralocorticoid hormones requires both extramitochondrial and intramitochondrial enzymes. Forexample, in the synthesis ofcortisol, pregnenolone must exit from the mitochondria to undergo conversions by 3p-hydroxysteroid dehydrogenase (a non-P450 microsomal enzyme) and by microsomal P450c17 and P450c21. The resulting product, 11-deoxycortisol, must then reenter the mitochondria for conversion to cortisol by P450cll (for review, see ref. 4). It is not clear how the steroidal intermediates are shuttled to the various cellular components, whether these components are closely aggregated in space, or whether these specific subcellular locations are required for the functioning of the steroidogenic pathways (for review, see ref. 5). However, the subcellular localization of enzymes has important consequences: the conversion of cholesterol to pregnenolone by mitochondrial P450scc appears to be the rate-limiting reaction in steroidogenesis because transport of cholesterol substrate into the mitochondria is slow, rather than because of inherent inefficiency of P450scc (14-16

Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants

“…The oligonucleotide primers used to produce the fusion proteins (oligonucletides 5-10) introduced the sequences required to create a hydrophilic linker peptide and provided the unique restriction sites needed to assemble the fusion constructs. The length and amino acid composition of the linker peptide are based on the hinge regions of naturally occurring multidomain proteins (16) as detailed previously (7,8,17). Initially, each construct was evaluated for its ability to produce RNA (Fig.…”

“…reductase domains used were from both mammalian (AdRed and OR) and bacterial (P450 BM-3) systems. Previously, we demonstrated that electron equivalents are passed ef ciently to cytochrome P450s only when they are linked to the proper electron transport protein (8). Thus, the presence of the other reductase domains was predicted to reduce the likelihood of producing a functional enzyme.…”

“…Since it was unclear whether either the heme or reductase domains would have suf cient sequences to interfere with dimer formation in wild-type nNOS, the heme and reductase domains were also linked together by a small hydrophilic peptide to form a heme-reductase fusion protein (heme-RedF). However, it was possible that heme-RedF would be active since previous studies linking cytochrome P450 proteins with their electron transport proteins had produced functional fusion enzymes (7,8,17). Thus, a set of fusion proteins that replaced the native nNOS reductase with the reductase domains from other electron transport systems was also constructed.…”

“…Previously, fusion proteins of P450scc linked to AdRed and OR had been constructed (7,8), and these plasmids served as the source of the cDNAs used to produce the heme-AdRed and heme-OR expression plasmids. Brie y, SpeI fragments containing either AdRed or OR were isolated from plasmids previously designated pF1 (7) and pF4 (8) and were used to replace the reductase domain in the pUCSF/heme-reductase plasmid. Subsequently the heme-AdRed and heme-OR cDNAs were subcloned into the pECE expression vector to produce the plasmids pECE/heme-AdRed and pECE/heme-OR, respectively.…”

Short term regulation of aldosterone secretion after stimulation and suppression experiments in mice

“…Early on it had been demonstrated that the acute stimulation of glucocorticoid output from the adrenal gland by ACTH is sensitive to protein synthesis inhibitors (Ferguson 1963). Further studies identified steroidogenic acute regulatory protein (StAR) to initiate the cascade of steroid biosynthesis with the translocation of cholesterol from the outer to the inner mitochondrial membrane (Black et al 1994, Clark et al 1994, Spat & Hunyady 2004. StAR expression is enhanced by ACTH and Ca 2C mediated stimuli such as angiotensin II (ANGII) and potassium (Spat & Hunyady 2004).…”