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.     

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.     

Studies on the immunochemical and functional similarities among iron-sulfur proteins involved in mitochondrial steroid hydroxylations

The effects of a goat anti-adrenodoxin immunoglobulin fraction on the NADPH- and NADH-dependent electron transport sequences in the mitochondria of steroidogenic tissues have been examined. The NADPH-cytochrome $\text{c}$ reductase activities of sonicated mitochondrial preparations derived from bovine adrenal cortex and rat adrenals and ovaries were inhibited in a similar manner in the presence of this antibody, while the inhibition of this activity in rat testicular mitochondrial preparations was less pronounced. The NADH-dependent reduction of cytochrome $\text{c}$ catalyzed by these mitochondrial preparations was not affected by the antibody. These results indicate that, while they may not be identical, the iron-sulfur proteins involved in mammalian mitochondrial steroid hydroxylations exhibit immunochemical and functional similarities.     

Isolation from bovine liver mitochondria of a soluble ferredoxin active in a reconstituted steroid hydroxylation reaction.

An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome $\text{c}$ reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (g_x = g_y = 1.935 and g_z = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P₄₅₀, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P₄₅₀ solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol.     

Kinetic differences between two interconvertible forms of fructose-1,6-bisphosphatase from Saccharomyces cerevisiae

Newly synthesized, [³⁵S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, 11β-hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [³⁵S]methionine-labeled 11β-hydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.     

Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

• 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
• 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
• 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450_scc.
• 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450_scc.

     

Immunohistochemical studies on electron transport proteins associated with cythochromes P-450 in steroidogenic tissues II. Microsomal NADPH-cytochrome c reductase in the rat adrenal

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum procued against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase techniques and an indirect fluorecent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

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.     

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.     

Immunohistochemical localization of adrenal ferredoxin and distribution of adrenal ferredoxin and cytochrome P-450 in the rat adrenal

Adrenal ferredoxin, the iron-sulfur protein associated with cytochromes P-450 in adrenocortical mitochondria, has been localized immunohistochemically at the light microscopic level in rat adrenals by employing rabbit antiserum to bovine adrenal ferredoxin in both an unlabeled antibody peroxidase-antiperoxidase method and an indirect fluorescent antibody method. When sections of rat adrenals were exposed to the adrenal ferredoxin antiserum in both procedures, positive staining for adrenal ferredoxin was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. Marked differences in the intensity of staining, however, where observed among the three cortical zones: the most intense staining being found in the zona fasciculata, less in the zona reticularis, and least in the zona glomerulosa. Furthermore, differences in staining intensity were also observed among cells within both the zona fasciculata and the zona reticularis. In agreement with these immunohistochemical observations, determinations of adrenal ferredoxin contents by electron paramagnetic resonance (EPR) spectrometry in homogenates prepared from capsular and decapsulated rat adrenals revealed that the concentration of adrenal ferredoxin in the zona glomerulosa was lower than that in the zona fasciculata-reticularis. Similar results were obtained when the contents of cytochrome P-450 were determined in capsular adn decapsulated rat adrenal homogenates. These observations indicate that adrenal ferrodoxin and cytochrome P-450 are not distributed uniformly throughout the rat adrenal cortex.     

Oxidation-reduction potentials of cytochromes P-450 and ferredoxin in the bovine adrenal: Their modification by substrates and inhibitors

The midpoint reduction potentials of the haem iron in bovine adrenal cytochrome P-450 and its associated iron-sulphur protein, adrenal ferredoxin, have been measured, using EPR spectroscopy to monitor the high and low spin ferric haem iron and reduced adrenal ferredoxin signals as a function of potential, in mitochondrial and microsomal suspensions.In mitochondria the high spin (substrate-bound) cytochrome P-450 showed single-component one-electron plots under most conditions; at pH 6.65 cholesterol side-chain cleavage cytochrome P-450 (P-450_scc) had a midpoint E_m = ?305 mV; at pH 8.0 11β-hydroxylase cytochrome P-450 (P-450_11β) had E_m = ?335 mV. Low spin cytochrome P-450 showed more complex titration curves under all conditions, which could be most simply interpreted in terms of two one-electron components with midpoint potentials approx. ?360 and ?470 mV, with varying intensities. During treatments that caused substrate binding, only the ?470 mV component was reduced in magnitude. On sonication and removal of adrenal ferredoxin, the ?470 mV low spin component was converted to higher potential. The potentials could also be altered by the cytochrome P-450 inhibitors aminoglutethimide and metyrapone. In the microsomes, a high spin component of cytochrome P-450 (E_m ≈ ?290 mV) was observed even at pH 8.0, suggesting the binding of an endogenous substrate, while the low spin P-450 showed a predominance of the ?360 mV component. The midpoint potential of membrane-bound adrenal ferredoxin under these various conditions was found to be ?248 mV ± 15 mV.     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Chemical cross-linking of ferredoxin to spinach thylakoids: Evidence for two independent binding sites of ferredoxin to the membrane

Ferredoxin has been chemically cross-linked to thylakoids by using N-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The membranes thus treated became able to photoreduce cytochrome c and to catalyze the NADPH-cytochrome c reductase reaction without adding exogenous ferredoxin. Preincubation of thylakoids with an antibody against ferredoxin-NADP⁺ reductase before carbodiimide treatment or removal of the reductase by mild trypsin treatment after the cross-linking reaction did not alter the cytochrome c photoreduction activity of the treated membranes. Two independent binding sites of ferredoxin to thylakoids are thus inferred: one site is shown to be the membrane-bound reductase, the second is suggested to be at the level of the photosystem I complex.     

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.     

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.     

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 side-chain cleavage, cytochrome P450, and iron-sulfur protein in human placental mitochondria.

Cytochrome P450 and the associated iron-sulfur protein have been characterized in human placental mitochondria by means of optical absorbance difference spectrophotometry and electron paramagnetic resonance spectrometry. These two proteins occur in a molar ratio of about 1:1 in human placental mitochondria, and the cytochrome P450 appears to be that form involved in cholesterol side-chain cleavage. Pregnenolone formation from endogenous mitochondrial cholesterol, as measured by radioimmunoassay, follows a biphasic time-course similar to the situation in other steroidogenic tissues. The specific activity of cholesterol side-chain cleavage, and the specific contents of cytochrome P450 and the iron-sulfur protein in the mitochondria, are 2- to 3-fold higher at term than in the 1st and 2nd trimesters. When expressed in terms of the cytochrome P450 content, the rate of pregnenolone formation is high, suggesting that cholesterol side-chain cleavage in human placenta is in an activated state.     

Purification and characterization of human placental ferredoxin

A ferredoxin-type iron-sulfur protein was isolated from human placenta mitochondria. The properties of the purified protein were very similar to those of adrenal ferredoxin (adrenodoxin), and immunological cross-reactivity with polyclonal antibodies to bovine adrenodoxin was observed. The N-terminal amino acid sequence and the visible absorption spectrum were identical to bovine adrenodoxin. The molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 13,500), however, is slightly smaller than that of adrenodoxin, and the C-terminal sequence is different. Human placental ferredoxin can substitute for bovine adrenodoxin in reactions reconstituted with bovine adrenal enzymes which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone.     

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.