Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase

The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K(m) of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K(m) of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K(m) for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.     

Immunochemical studies on adrenal ferredoxin: Involvement of adrenal ferredoxin-like iron-sulfur proteins in the cholesterol side-chain cleavage reaction of mammalian steroidogenic tissues

The possible immunochemical and functional similarities existing among adrenal ferrdoxin-like iron-sulfur proteins present in the mitochondria of mammalian steroidogenic tissues have been examined by employing a goat antibody produced against homogeneous bovine adrenal ferredoxin. This antibody was found to inhibit the NADPH-cytochrome c reductase and cholesterol side-chain cleavage activities catalyzed by mitochondria prepared from rat adrenals, rat ovaries and the testes of rats which had been treated with human chorionic gonadotropin. No inhibition of the NADH-dependent reduction of cytochrome c catalyzed by these mitochondria was observed in the presence of the anti-adrenal ferredoxin. These results demonstrate that adrenal ferredoxin and the comparable iron-sulfur proteins of ovarian and testicular mitochondria are immunochemically similar and are required for the cholesterol side-chain cleavage reaction occurring in these tissues. Although a precipitin reaction was observed upon double diffusion of the anti-adrenal ferredoxin against human term placental mitochondria, no inhibition of either the NADPH-cytochrome c reductase or the cholesterol side-chain cleavage⁴ activity catalyzed by preparations of these mitochondria was observed in the presence of the antibody. These results indicate that the iron-sulfur protein present in human placental mitochondria at term differs immunochemically from other mammalian adrenal ferredoxin-like iron-sulfur proteins.     

Purification and immunological characterization of human placental mitochondrial cytochrome P-450

Human placental mitochondrial cytochrome P-450 was purified to electrophoretic homogeneity by hydrophobic, anion exchange and cation exchange column chromatography. The specific content of the purified protein was 15.7 nmol/mg protein and it showed a single band mol. wt 48,000 D in SDS-gel electrophoresis. When reconstituted with bovine adrenal adrenodoxin reductase and adrenodoxin it converted cholesterol to pregnenolone (cholesterol side-chain cleavage activity, CSCC) at the rate of 1 pmol/min/pmol P-450. Antibodies against the purified protein were raised in rabbits. Inhibition studies demonstrated 85% inhibition of placental CSCC activity at an antibody/protein ratio of 10:1. Placental microsomal aromatase activity was inhibited by 47% at the same antibody/protein ratio. The antibody inhibited bovine mitochondrial CSCC activity by 87% at the same antibody/protein ratio. Placental microsomal 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities were not significantly inhibited by the antibody. The results indicate that the purified protein catalyzes cholesterol side-chain cleavage reaction, human placental microsomal aromatase and bovine adrenal mitochondrial P-450scc may share common antigenic determinants with placental P-450scc, but the placental microsomal xenobiotic-metabolizing cytochrome(s) is (are) distinctly different.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.     

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates

The side-chain cleavage of cholesterol by cytochrome P-450_scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3β-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450_scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450_scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20-hydroxycholesterol, 22R-hydroxycholesterol and 20,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450_scc occur at approximately the same rate.     

Cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria and human placenta: immunochemical properties and structural characteristics

An immunochemical comparison of components of cholesterol side chain cleavage system from bovine adrenocortical and human placental mitochondria has been carried out. Antibodies against cytochrome P-450scc, adrenodoxin reductase and adrenodoxin from bovine adrenocortical mitochondria were shown to cross-react with corresponding antigens of human placental mitochondria. A highly sensitive immunochemical method for cytochrome P-450scc determination has been developed. Limited proteolysis of cytochrome P-450scc of human placental mitochondria was studied, and the products of trypsinolysis were identified using antibodies against cytochrome P-450scc and fragments of its polypeptide chain: F1, F2 and F3. Immunochemical relatedness of ferredoxins from bovine adrenocortical and human placental mitochondria allowed one to develop a fast and efficient method for cytochrome P-450scc purification from human placental mitochondria by affinity chromatography on adrenodoxin-Sepharose.     

Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.     

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.     

Side-chain cleavage of cholesterol sulfate by ovarian mitochondria

Mitochondria isolated from porcine corpora lutea and from the luteinized ovaries of gonadotropin-treated immature rats were found to efficiently cleave the side-chain of cholesterol sulfate to produce 3 beta-hydroxy-5-pregnen-20-one sulfate (pregnenolone sulfate). When mitochondria were preincubated with cholesterol sulfate, the time-course for the side-chain cleavage of cholesterol sulfate was biphasic. With 200 microM cholesterol sulphate, the initial rate of the reaction was the same as that observed for 25-hydroxycholesterol. This rate was not increased when both cholesterol sulfate and 25-hydroxycholesterol were incubated together. The rate of side-chain cleavage by isolated mitochondria supplied with 75 microM cholesterol sulfate as substrate was inhibited by 97% by aminoglutethimide, a specific inhibitor of cytochrome P-450scc. The slow phase of side-chain cleavage of cholesterol sulfate appeared to be limited by the rate of substrate movement to the mitochondrial site of the reaction. Cholesterol sulfate translocation rates were however up to 8 times greater than those observed for cholesterol when equivalent concentrations of the two substrates were added to the mitochondria. We conclude that cholesterol sulfate is a better substrate than cholesterol for side-chain cleavage by isolated mitochondria and that both reactions are catalysed by the same cytochrome P-450scc enzyme.     

Human adrenodoxin: cloning of three cDNAs and cycloheximide enhancement in JEG-3 cells

Adrenodoxin is an iron-sulfur protein serving as an electron transport intermediate for two mitochondrial steroidogenic cytochromes P450. We have cloned and sequenced three human adrenal adrenodoxin cDNAs. The longest 5'-untranslated region was 131 bases long, and the coding sequences, identical in all three clones, predict a preprotein of 180 amino acids. The 3'-untranslated regions were 235, 596, and 776 bases long due to the presence of alternate polyadenylation sites. RNA transfer blots showed multiple size species of adrenodoxin mRNA consistent with finding multiple polyadenylation sites. Similar sized cross-hybridizing RNA species are found abundantly in the adrenal and testis and to a lesser degree in RNA from human fetal brain, spleen, placenta, kidney, liver, and intestine, as well as in cultured fibroblasts, suggesting the same or a very similar iron-sulfur protein is found in mitochondria of nonsteroidogenic tissues. JEG-3 cells, a transformed progesterone-producing line of trophoblastic origin, accumulate mRNAs for cytochrome P450scc (the mitochondrial cholesterol side-chain cleavage enzyme), adrenodoxin, and the fos oncogene when stimulated with 8-bromo-cyclic AMP. Addition of actinomycin D to such cultures blocked cAMP-induced accumulation of mRNAs for cytochrome P450scc and adrenodoxin. Addition of cycloheximide or puromycin to such cultures substantially reduced basal levels and markedly attenuated the cAMP-induced accumulation of cytochrome P450scc mRNA, but augmented the accumulation of adrenodoxin and fos mRNAs in additive and multiplicative fashions, respectively. These data indicate that the cAMP-induced synthesis of the steroidogenic machinery is not wholly dependent on cycloheximide-sensitive protein mediators.     

Immunochemical evidence for the involvement of adrenal ferredoxin in the side-chain cleavage of 20α-hydroxycholesterol

A goat antibody produced against homogeneous bovine adrenal ferrodoxin has been employed to study the involvement of this iron-sulfur protein in the side-chain cleavage of 20α-hydroxycholesterol catalyzed by a soluble fraction, supernatant S₁, prepared from sonicated bovine adrenocortical mitochondria. When added to this supernatant, the antibody inhibited the side-chain cleavage of 20α-hydroxycholesterol as well as the side-chain cleavage of cholesterol, the 11β-hydroxylation of deoxycorticosterone, and the NADPH-dependent reduction of cytochrome $\text{c}$. These results demonstrate that, similar to the NADPH-cytochrome $\text{c}$ reductase and both the cholesterol side-chain cleavage and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the side-chain cleavage of 20α-hydroxycholesterol.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

The insulin-like growth factor, somatomedin C, induces the synthesis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in ovarian cells

The actions of insulin and somatomedin C (insulin-like growth factor I) on cholesterol side-chain cleavage activity and the synthesis of cytochrome P-450scc and adrenodoxin were investigated in primary cultures of swine ovarian (granulosa) cells. Nanomolar concentrations of pure human somatomedin C stimulated biosynthesis of progesterone and 20 alpha-hydroxypregn-4-en-3-one. Moreover, in the presence of exogenous sterol substrate for cholesterol side-chain cleavage, somatomedin C significantly enhanced pregnenolone biosynthesis in a time- and dose-dependent manner. This augmentation of functional cholesterol side-chain cleavage activity was accompanied by a dose-dependent (2-16-fold) increase in [35S]methionine incorporation into specific immunoprecipitable cytochrome P-450scc and adrenodoxin. Micromolar concentrations of insulin (but not proinsulin or desoctapeptide) also induced synthesis of cholesterol side-chain cleavage constituents by 4-7-fold. These results demonstrate that an insulin-like growth factor, somatomedin C, exerts discrete differentiating effects on ovarian cells characterized by increased synthesis of immunospecific cytochrome P-450scc and adrenodoxin. Thus, we infer that somatomedin C may serve a critical role in the differentiation of steroidogenic cells in the mammalian ovary.     

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.     

Ultrastructural localization of cytochrome P-450scc in the bovine placentome using protein A-gold technique

We have previously reported that the steroidogenic activity of the bovine placentome is stimulated by a calcium-mediated, cyclic nucleotide-independent mechanism and that this steroidogenesis is limited by the availability of sterol substrate to the side-chain cleavage enzyme. We have recently established that the antibody against bovine adrenal cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450scc) can be used to specifically detect P-450scc in both bovine placentome and corpus luteum. In the present study, we used an immunogold technique to localize the P-450scc in the bovine placentome by electron microscopy. The mononucleate cell of the cotyledon showed both giant and normal-sized mitochondria, with the latter, predominating. Both mitochondrial types found in the mononucleate cells clearly displayed gold particles located on the cristae; in contrast, these particles were absent in the binucleate cells. It is worth noting that giant mitochondria were found exclusively in the placental mononucleate cells in both the fetal and maternal sites but not in the binucleate cells. These findings suggest that the cholesterol side-chain cleavage enzyme is present in bovine cotyledon cells, primarily in mononucleate cells. The variations in P-450scc immunoreactivity among different cells of the placenta are suggestive of different steroidogenetic capacities of the cells.     

The presence of an adrenodoxin-like ferredoxin and cytochrome P-450 in brain mitochondria.

An iron-sulfur protein has been isolated from bovine brain mitochondria and purified 200-fold. The optical spectrum (peaks at 412 and 455 nm which disappear upon reduction) and the EPR spectrum (g values at 1.94 and 2.02) were typical for a ferredoxin. In reconstitution experiments, the protein could replace adrenodoxin in the cholesterol side chain cleavage reaction. The additional detection of cytochrome P-450 in brain mitochondria indicates that the isolated ferredoxin is part of a cytochrome P-450-dependent hydroxylation system.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Cholesterol sulfate is a naturally occurring inhibitor of steroidogenesis in isolated rat adrenal mitochondria

We previously reported (Lambeth, J. D., Xu, X. X., and Glover, M. (1987) J. Biol. Chem. 262, 9181-9188) that exogenously added cholesterol sulfate inhibits the conversion of cholesterol to pregnenolone in isolated adrenal mitochondria, and does so by affecting intramitochondrial cholesterol movement but not its subsequent metabolism to pregnenolone by cytochrome P-450scc. We now report that a major kinetic component of the inhibition is noncompetitive with respect to cholesterol, consistent with an allosteric effect at a site other than the substrate binding site of cytochrome P-450scc. We now also report that cholesterol sulfate is present as an endogenous compound in preparations of adrenal mitochondria. Its content varied from 0.05 to 0.8 nmol/mg protein. Cholesterol sulfate level correlated inversely with the mitochondrial cholesterol side-chain cleavage activity. Endogenous cholesterol sulfate thus appeared to account for the variable rates of pregnenolone synthesis which were seen in different mitochondrial preparations. Cholesterol sulfate was metabolized to pregnenolone sulfate by a mitochondrial side-chain cleavage system, but proved to be a relatively poor substrate for an extramitochondrial steroid sulfatase activity present in adrenal cortex. Confirming a role as a naturally occurring inhibitor, removal of endogenous mitochondrial cholesterol sulfate by metabolism to pregnenolone sulfate correlated with a 3-fold activation of cholesterol side-chain cleavage. We suggest that cholesterol sulfate functions in steroidogenic tissues to regulate the magnitude of the steroidogenic response.