Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Purification and comparative characterization of cytochrome P-450scc from porcine adrenocortical mitochondria.

Cytochrome P-450scc (cholesterol side-chain cleavage enzyme) was purified from porcine adrenocortical mitochondria. 2. The purified cytochrome P-450scc was found to be homogeneous on SDS-polyacrylamide gel electrophoresis. 3. The heme content of the purified enzyme was 20.6 nmol/mg protein. 4. The enzymatic activity of the reconstituted cytochrome P-450scc-linked monooxygenase system amounted to 7.8 nmol of pregnenolone formed per nmole of P-450 per minute, with cholesterol as a substrate. 5. The amino acid sequence of the amino-terminal region of the cytochrome P-450scc and the amino acid residue at the carboxyl terminal were determined and compared with those of other mammalian cytochromes P-450scc.     

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.     

Use of cytochrome P-450scc to measure cholesterol-lipid interactions

The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability in both micellar and vesicular environments. By use of this approach, in conjunction with titration of putative cholesterol binding species, a tight, approximately equimolar complex of cholesterol and digitonin was demonstrated. Sphingomyelin (SM) (both the synthetic N-palmitoyl and bovine brain forms) gave sigmoidal titration curves, suggesting a cooperative interaction between this lipid and cholesterol. The interaction of bovine brain glycerolipids and cholesterol was weaker than that of SM and showed no cooperativity. The importance of the phospholipid head group in these interactions was established by the differences in the ability of synthetic 1-palmitoyl-2-oleoylphosphatidylcholine, -phosphatidylethanolamine, and -phosphatidylserine to affect cholesterol availability. Comparison of these results with those of the bovine brain phospholipids indicates that the acyl chain composition of these molecules is also important to these interactions. Titrations of SM in phospholipid vesicles containing cytochrome P-450scc and different types of phosphatidylcholine established that the SM-cholesterol interactions also occur in a bilayer membrane. This study demonstrates that the association of cholesterol with cytochrome P-450scc is inhibited by concentrations of SM commonly found in biological membranes. Therefore, such cholesterol-lipid interactions can potentially affect the function of membrane enzymes.     

Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.     

Primary structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria and some aspects of its functioning on a structural level

The primary structure of the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) from bovine adrenocortical mitochondria has been determined. At the initial stage an exhaustive chymotryptic digestion of carboxymethylated P-450scc was performed, and the amino acid sequence of 66 peptides was determined. At the second stage an investigation of the amino acid sequence of individual fragments I (Mr 29 800) and II (Mr 26 600) of the limited trypsinolysis of P-450scc was carried out. Fragment I was digested with trypsin, Staphylococcus aureus V8 proteinase and thermolysin; fragment II was cleaved with trypsin and S. aureus V8 proteinase. In addition, the amino acid sequence of some CNBr peptides of P-450scc has been investigated. The primary structure of cytochrome P-450scc determined with protein chemistry methods proved the multistage cholesterol transformation to pregnenolone to be catalyzed by a single species of cytochrome P-450scc which consists of 481 amino acids. The results from protein sequencing of P-450scc are in good agreement with those obtained recently from nucleotide sequencing. The localization of peptide bonds cleaved under limited proteolysis of P-450 with trypsin to fragments I and II, I and III (Mr 16 800) is presented. It is shown that the transformation of P-450scc to P-420 is accompanied by the appearance of an additional trypsin-sensitive peptide bond in the N-terminal part of P-450scc.     

Polyphosphorylated glycerolipids mimic adrenocorticotropin-induced stimulation of mitochondrial pregnenolone-synthesis.

As with adrenocorticotropin pretreatment in vivo, addition of cardiolipin in vitro enhances adrenal mitochondrial pregnenolone synthesis and apparent binding of cholesterol to cytochrome P-450scc. These effects are relatively specific for glycerolipids containing two or more phosphate radicals in the polar head group, and changes in such phospholipids or comparably acting substances may play a role in mediating adrenocorticotropin- or other hormone-induced effects on membrane-associated enzymes.     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

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.     

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Phospholipids, sterol carrier protein2 and adrenal steroidogenesis

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein2 (SCP2) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of phosphatidylcholine, phosphatidylinositol, or the 4'-phosphate and the 4',5'-diphosphate derivatives of phosphatidylinositol were without effect. With the adrenal plasma membrane preparation, none of the added phospholipids affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact mitochondria, only cardiolipin, among the various phospholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 microM), the stimulatory effect was only half that observed with 0.7 microM SCP2, and the two effectors were not synergistic. SCP2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450scc, had no effect on distribution of mitochondrial membrane cholesterol.     

Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc

Covalent modification of cytochrome P-450scc (purified from bovine adrenocortical mitochondria) with pyridoxal 5'-phosphate (PLP) was found to cause inhibition of the electron-accepting ability of this enzyme from its physiological electron donor, adrenodoxin, without conversion to the "P-420" form. Reaction conditions leading to the modification level of 0.82 and 2.85 PLP-Lys residues per cytochrome P-450scc molecule resulted in 60% and 98% inhibition, respectively, of electron-transfer rate from adrenodoxin to cytochrome P-450scc (with beta-NADPH as an electron donor via NADPH-adrenodoxin reductase and with phenyl isocyanide as the exogenous heme ligand of the cytochrome). It was found that covalent PLP modification caused a drastic decrease of cholesterol side-chain cleavage activity when the cholesterol side-chain cleavage enzyme system was reconstituted with native (or PLP-modified) cytochrome P-450scc, adrenodoxin, and NADPH-adrenodoxin reductase. Approximately 60% of the original enzymatic activity of cytochrome P-450scc was protected against inactivation by covalent PLP modification when 20% mole excess adrenodoxin was included during incubation with PLP. Binding affinity of substrate (cholesterol) to cytochrome P-450scc was found to be increased slightly upon covalent modification with PLP by analyzing a substrate-induced spectral change. The interaction of adrenodoxin with cytochrome P-450scc in the absence of substrate (cholesterol) was analyzed by difference absorption spectroscopy with a four-cuvette assembly, and the apparent dissociation constant (Ks) for adrenodoxin binding was found to be increased from 0.38 microM (native) to 33 microM (covalently PLP modified).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of pregnenolone synthesis by cytochrome P-450scc in mitochondria from porcine corpora lutea and granulosa cells of follicles

Substrate turnover rates by cytochrome P-450scc were measured in mitochondria isolated from corpora lutea and granulosa cells of follicles. Hydroxycholesterol substrates were added to the mitochondria to test the degree of saturation of the cytochrome with endogenous cholesterol during pregnenolone synthesis. 25-Hydroxycholesterol proved unsuitable for this since it was converted into pregnenolone with a maximum velocity of only 25% of that for cholesterol. 20 alpha-Hydroxycholesterol was found to be suitable providing correction was made for the one less hydroxylation required to convert this substrate into pregnenolone, compared to cholesterol. Mitochondria isolated from large follicles and corpora lutea displayed biphasic time courses for pregnenolone synthesis from endogenous cholesterol with a rapid phase lasting for 2-4 min and a slow phase which was linear for at least 30 min. Only a single rapid phase was observed for these mitochondria in the presence of 20 alpha-hydroxycholesterol. From the degree of stimulation of the substrate turnover rate by this steroid, it was concluded that the endogenous cholesterol concentration was saturating during the fast phase for large follicles but subsaturating in luteal mitochondria. Time courses for pregnenolone synthesis by mitochondria isolated from granulosa cells of small and medium follicles were linear for 30 min and gave a substrate turnover rate of 16-18 mol of steroid/min/mol of cytochrome P-450scc, similar to the turnover rates under saturating substrate conditions determined for large follicles and corpora lutea. The substrate turnover rate for cytochrome P-450scc in medium follicles was not increased by the addition of 20 alpha-hydroxycholesterol, indicating that the cholesterol concentration in the steroidogenic pool of these mitochondria was saturating and remained so over the 30-min duration of the incubation. It is therefore unlikely that gonadotropin stimulation of granulosa cells of small to medium follicles could acutely regulate pregnenolone synthesis by increasing the rate of transfer of cholesterol into a steroidogenic pool. This study shows that as the cytochrome P-450scc concentration in porcine ovarian mitochondria increases during follicular growth and luteinization there is a decrease in the fractional saturation of the cytochrome with cholesterol.     

Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Interaction of a spin-labelled cholesterol derivative with the cytochrome P-450scc active site

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km = 33 microM, Vmax = 0.32 min-1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 microM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd = 5.9 microM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd = 1.9 microM). This finding is in accord with a rotational correlation time of about 10(-7) s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450scc displays the ESR g-values gx = 2.404/2.456, gy = 2.245 and gz = 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450scc. Our data indicate that the stereospecificity of the cytochrome P-450scc side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6-1.0 nm from the heme moiety.