Mitochondrial P450 Research Articles

Molecular cloning and nucleotide sequence of DNA of mitochondrial cytochrome P-450(11 beta) of bovine adrenal cortex.

cDNA clones of the mRNA for bovine adrenal cytochrome P-450(11 beta) were isolated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11 beta), which consisted of 503 amino acids (Mr: 57,924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11 beta). molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11 beta) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11 beta) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11 beta) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11 beta) and P-450(SCC) was 36%, which is higher than the values between P-450(11 beta) and various microsomal P-450s. An alignment of P-450(11 beta) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hydropathy profiles of P-450(11 beta) and P-450(SCC) were very similar. A definite difference was noticed at the NH2-terminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydrophobic sequence consisting of about 20 amino acids, whereas mitochondrial type had an extension peptide containing many positively changed amino acids.

Cholesterol metabolism in estrogen-sensitive progestin synthesis by rabbit corpus luteum.

To learn whether either reduced de novo cholesterol synthesis and/or altered cholesteryl ester metabolism is responsible for the deficient progestin production induced by estrogen withdrawal from pseudopregnant rabbits, we measured the luteal activity of three enzymes: 1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (the rate-limiting step in de novo cholesterol synthesis), 2) cholesteryl ester hydrolase, and 3) acyl coenzyme A:cholesterol acyltransferase (ACAT) in estrogen-stimulated and estrogen-deprived rabbits. The only change in the activity of these enzymes and of the enzyme NADPH-cytochrome c reductase (a microsomal marker enzyme) after estrogen capsule removal for 12 or 24 h was a 30% decrease in HMG-CoA reductase activity after 24 h. The decrease in HMG-CoA reductase activity was not accompanied by a detectable change in either the content or localization of cellular free cholesterol. Previous data from our laboratory have demonstrated that 24 h of estrogen deprivation has no effect on inner mitochondrial membrane P-450 side-chain cleavage activity (a rate-limiting step in the conversion of cholesterol to steroid hormones). These data, and our earlier finding that estrogen deprivation leads to accumulation of cholesteryl ester in the luteal cells, indicate that estrogen maintains rabbit luteal progestin production by stimulating the transfer of cytoplasmic cholesterol to the active site of P-450 side-chain cleavage on the inner mitochondrial membrane.

P-450 11β-Dependent conversion of androgen to estrogen, the aromatase reaction

Purified bovine adrenal P-450 11β has been shown to act as an aromatase which catalyzes conversion of 19-oxoandrostenedione to estrone. No conversions took place when any one of the required components such as NADPH, NADPH:adrenodoxin reductase, adrenodoxin and P-450 11β was omitted from the complete reconstituted system. P-450scc, another mitochondrial P-450 obtained from adrenal cortex, did not substitute for the P-450 11β in the aromatase reaction. These results show that P-450 11β is able to catalyze a series of reaction which can generate adrenal estrogen through androstenedione and its 19-hydroxy- and 19-oxo-derivatives. The P-450 11β-dependent reaction appears to be quite different from the placental aromatase reaction in that the latter is catalyzed by a microsomal P-450.

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase.

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.

Interaction of bovine renal mitochondrial cytochrome P-450 with antioxidants

The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me 2SO), protocatechuic acid (PCA), and P-450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D 3 1α- and 24-hydroxylase activities in cultured bovine proximal tubule cells. The slowing of the loss of hydroxylase activities by antioxidants was increased by culturing cells in 5% O 2 vs 19% O 2. These same antioxidants also directly inhibited 1α- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, K i 's for inhibition of both hydroxylases were equal, ED 50's for stabilization of both hydroxylase activities were equal, and K i 's and ED 50's were not significantly different. These antioxidants prevented tert-butylhydroperoxide ( tert-BOOH)-mediated proximal tubule cell death at concentrations i.e., 0.1 m m, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H 2SeO 3, uric acid, and trolox c gave slight stabilization of hydroxylase activities without inhibiting hydroxylase activities. Singly, these antioxidants did not stabilize or directly inhibit hydroxylase activities. This antioxidant combination augmented BHA- or BHT-mediated stabilization of both hydroxylase activities independent of any effects on inhibition. But the most potent antioxidants which acted to stabilize hydroxylase activities in culture also directly acted to inhibit hydroxylase activities. Antioxidant effects were additive for both inhibition and stabilization of hydroxylase activities. Stabilization of hydroxylase activities was dissociated from inhibition in the presence of maximal FA, CA, and BHA or FA, CA, and BHT combinations. Bovine renal mitochondrial cytochrome P-450 levels decreased in cultured bovine proximal tubule cells to nondetectable levels by 8 days in culture. When cultures were treated with BHA and BS, mitochondrial P-450 levels were almost twofold greater than in untreated controls. Percentage changes in mitochondrial P-450 levels closely paralleled percentage changes in hydroxylase activities elicited by antioxidant treatment regimes. Antioxidants which were effective inhibitors of hydroxylase activities in cultured bovine proximal tubule cells were also effective in inhibiting hydroxylase activities in isolated proximal tubule mitochondria, supplemented with a NADPH-generating source. K i 's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria. Antioxidants were shown to inhibit hydroxylase activities, in isolated mitochondria, in a competitive manner by analysis of experiments which varied substrate and antioxidant concentrations. The importance of antioxidants to the maintenance of vitamin D 3 metabolism in vitro is discussed as is relevance of these findings to maintenance of in vivo renal mitochondrial vitamin D 3 metabolism.

Subcellular distribution of cytochrome P-450 in the brain

The subcellular distribution of the monoogynase complexes in the brain was studied by using subcellular fractionation and characterization of these fractions by marker enzymes. Cytochrome P-450 was found to be mainly localized in both synaptic and non-synaptic mitochondria; only a small quantity of enzyme was also found in the microsomal fraction. Peeling off the outer membrane of mitochondria showed that the protein was retained in the inner membrane fraction. A comparative study among some other species confirmed the mitochondrial prevalence of cerebral cytochrome P-450. A partial purification of the rat brain mitochondrial P-450 was obtained.

Inhibition of steroidogenic cytochrome P-450 enzymes in rat testis by ketoconazole and related imidazole anti-fungal drugs

Ketoconazole, an imidazole antifungal drug, has previously been shown to diminish testosterone and cortisol production in patients as well as rat and mouse cells in vitro. Inhibition of adrenal mitochondrial cytochrome P-450 enzymes was demonstrated. In this study we tested several imidazole antifungal drugs and examined the individual steps in testicular steroidogenesis to determine which enzymes in the androgen pathway were blocked. In addition, we studied 25-hydroxyvitamin D 24-hydroxylase activity in cultured pig kidney cells (LLC-PK 1) to assess a mitochondrial P-450 enzyme in another organ. All imidazoles tested inhibited both total testosterone production and 24-hydroxylase activity but the relative potencies differed. We next studied the individual testicular enzymatic steps between cholesterol and testosterone. Ketoconazole inhibited cholesterol-side-chain-cleavage enzyme (mitochondrial) and C-17,20 lyase (microsomal). The three inhibited enzymes (two testicular and one renal) are all P-450 cytochromes. Testicular 17-hydroxylase, also a P-450 cytochrome, was not inhibited even at high doses of ketoconazole. This is an interesting finding because the testicular hydroxylase and lyase have been shown to be a single protein. Non-cytochrome P-450 enzymes in the androgen pathway were not inhibited. The results demonstrate that several imidazole antifungal drugs all inhibit both microsomal and mitochondrial cytochrome P-450 enzymes in multiple organs.

Liver alcohol oxidizing systems and gluconeogenic enzyme activities after long term ethanol application in cold exposed guinea pigs.

The effects of 4-weeks ethanol application (20% ethanol, w/w, 2 g X kg-1 on the alcohol oxidizing systems and gluconeogenic enzyme activities of the liver in guinea pigs kept in the cold (+4 degrees C) and at room temperature (+20 degrees C) were studied. The controls were guinea pigs reared at room temperature or in a cold environment without ethanol. The study showed a significant increase (1.5-fold) in liver microsomal cytochrome P-450 after chronic ethanol treatment at room temperature, but not in a cold environment. Microsomal NADPH oxidase activity did not significantly change in any group. Ethanol treatment in a cold environment resulted in a significant increase in liver mitochondrial cytochromes, aa3 and c+c1, and at room temperature in cyt aa3. The activities of total liver homogenate alcohol dehydrogenase or catalase did not change after chronic ethanol treatment. The activity of liver fructose-1.6-diphosphatase showed a significant ethanol induced decrease at room temperature, an effect not observed in the cold environment. Ethanol increased glucose-6-phosphatase activity in the cold, but not at room temperature. In conclusion, the stimulation of liver mitochondrial cytochromes and microsomal cyt P-450 as a consequence of chronic ethanol treatment indicated an increased oxidation capacity for ethanol. The stimulation of glucose-6-phosphatase in a cold environment might be responsible for increasing glucose for heat production after chronic ethanol treatment in cold adapted animals.

25-hydroxyvitamin D hydroxylation. Evidence for a dioxygenase activity of solubilized renal mitochondrial cytochrome P-450.

Upon solubilization and partial purification, renal mitochondrial P-450 catalyzes both the 1 alpha- and 24-hydroxylations of 25-hydroxyvitamin D3 in the absence of NADPH. Neither 1 alpha,25-dihydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 is further metabolized by the enzyme. Under similar conditions, P-450 obtained from hepatic microsomes or adrenal mitochondria is inactive as a 25-hydroxyvitamin D3 hydroxylase. Both hydroxylations are heat-sensitive and are inhibited by 1 alpha,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin, 25,26-dihydroxyvitamin D3, EDTA, menadione, dithiothreitol, and cadmium but not by carbon monoxide. Cumene hydroperoxide does not facilitate either hydroxylation and no lipid peroxides can be detected in the system either prior to or after incubation with the substrate. Based on these data, it is proposed that during these hydroxylations P-450 is acting as a dioxygenase. The first step in the sequence of reactions is postulated to be a P-450-catalyzed formation of a hydroperoxide at carbon 1 of a substrate molecule. This is followed by the 25-hydroxyvitamin D hydroperoxide-supported, P-450-catalyzed hydroxylation of another molecule of substrate at either the 1 alpha or the 24 position. During the cycle 1 alpha,25-dihydroxyvitamin D3 is also generated from the hydroperoxide. From this mechanism, it can be concluded that one form of P-450 is capable of catalyzing the production of both 1 alpha,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

Ketoconazole blocks adrenal steroidogenesis by inhibiting cytochrome P450-dependent enzymes.

Ketoconazole has recently been shown to interfere with steroidogenesis in patients and rat in vitro systems. In this study we attempted to elucidate the site of inhibition in the adrenal gland. Although ketoconazole impaired adrenocorticotropic hormone stimulated cyclic (c)AMP production, dibutyrl cAMP addition did not bypass the steroidogenic blockade indicating that the critical ketoconazole-inhibited step was distal to cAMP. Addition of radiolabeled substrates to isolated adrenal cells and analysis of products by high performance liquid chromatography demonstrated a ketoconazole block between deoxycorticosterone (DOC) and corticosterone. This 11-hydroxylase step is carried out by a P450-dependent mitochondrial enzyme. No restriction of progesterone or pregnenolone conversion to DOC was detected, steps carried out by non-P450-dependent microsomal enzymes. Inhibition of cholesterol conversion to pregnenolone by mitochondrial fractions indicated a second block at the side chain cleavage step, another mitochondrial P450-dependent enzyme. Adrenal malate dehydrogenase, a non-P450-dependent mitochondrial enzyme was not inhibited while renal 24-hydroxylase, a P450-dependent mitochondrial enzyme in another organ, was blocked by ketoconazole. We conclude that ketoconazole may be a general inhibitor of mitochondrial P450 enzymes. This finding suggests that patients receiving ketoconazole be monitored for side effects relevant to P450 enzyme inhibition. Further, we raise the possibility that this drug action may be beneficially exploited in situations where inhibition of steroidogenesis is a therapeutic goal.

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of C57B1 6 mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.

Solubilization and partial purification of human placental cytochromes P-450

Human placental mitochondrial and microsomal cytochrome P-450 (P-450) was solubilized with sodium cholate in the presence of Emulgen 911 and the solubilized preparations purified by phenyl-Sepharose and DEAE-cellulose column chromatography. The final preparations exhibited specific activities between 3.0 and 7.0 nmol P-450/mg protein implying a 30–115-fold purification over the starting material. Androstenedione exhibited type I spectral interaction with the microsomal P-450 and pregnenolone a reverse type I spectrum with mitochondrial P-450. Hydrophobic column chromatography proved to be a rapid and efficient initial purification step for placental P-450s.

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the in vitro products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40-50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20-30 sec of incubation with deoxycholate. The content of RNA, phospholipids, and cytochrome b5 in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.

Implication of ligand modified spectra of cytochrome P-450 associated with pregnenolone synthesis in mitochondria from corpus luteum.

The implications of ligand modified spectra of cytochrome P-450 in mitochondria from corpus luteum are considered. Mitochondria from bovine corpus luteum contain a single cytochrome P-450 which oxidizes cholesterol to pregnenolone and isocaproaldehyde. These mitochondria and the cytochrome P-450 purified from these mitochondria yield Type I spectral changes with substrates, reverse Type I spectral changes with certain steroid substrates, steroid products and unrelated steroid ligands. Nitrogenous ligands yield Type II spectral changes. Mitochondrial and purified cytochrome P-450 preparations are equivalent in this study. The inhibitory effects on the cholesterol monooxygenase are directly related to the spectral changes induced by Type II ligands. Lastly, it is suggested that a similar relationship exists with reverse Type I ligands.

Adrenal Mitochondrial Cytochrome P-450 and Cholesterol Side Chain Cleavage Activity: DIFFERENCES IN THE RESPONSE OF THE ZONA GLOMERULOSA AND ZONA FASCICULATA-RETICULARIS TO ADRENOCORTICOTROPIC HORMONE AND ITS WITHDRAWAL

Abstract A comparison of the effects of adrenocorticotropic hormone (ACTH) on the cholesterol side chain cleavage system of zona glomerulosa and zona fasciculata-reticularis tissue of the rat adrenal is presented. Following hypophysectomy, the rate of pregnenolone formation from endogenous cholesterol in mitochondria from zona fasciculata-reticularis was lower than in mitochondria from zona glomerulosa. Also, the proportion of high spin, cholesterol-bound side chain cleavage cytochrome P-450 was smaller, as measured by spectrophotometry and electron paramagnetic resonance spectroscopy. Treatment with ACTH brought about an increase in the rate of cholesterol side chain cleavage and in the proportion of high spin, cholesterol-bound side chain cleavage cytochrome P-450 in mitochondria isolated from both zones. However, the percentage increase found in the zona fasciculata reticularis was much greater than in the zona glomerulosa. It is concluded that although both zones are affected by ACTH, the cholesterol side chain cleavage system of the zona fasciculata-reticularis is much more sensitive than that of the zona glomerulosa to this hormone.

Binding of prostaglandins and cytochrome P-450

Prostaglandins F 1α, E 1 and E 2, as well as their fatty acid precursors were found to induce resp. Type II and Type I spectral changes with cytochrome P-450 obtained from bovine adrenal microsomes. No such spectra were observed with the mitochondrial P-450 fractions. The binding affinities of the prostaglandins, as measured by their apparent dissociation constants K s, are of the same order of magnitude as the values calculated for the endogenous steroid hormones progesterone, 17α-hydroxyprogesterone and estradiol. These constants were affected by the presence of carbon monoxide. The above original observations could serve as an indication that cytochrome P-450 may play a role in either the biosynthesis or metabolism of prostaglandins.

Studies on the biosynthesis of 18-oxygenated steroids from exogenous corticosterone by domestic duck ( Anas platyrhynchos) adrenal gland mitochondria

The transformation of exogenous, isotopically labelled corticosterone to 18-hydroxycorti-costerone and aldosterone by domestic duck ( Anas platyrhynchos) adrenal gland mitochondria was studied. The mitochondrial 18-oxygenating system was NADPH dependent. K +, Na +, Ca 2+ and Mg 2+ were necessary for maximal enzymatic activity. The enzyme present in 1 mg of mitochondrial protein became saturated with 13.2 βM of substrate (13.8 μg). Under saturation conditions the maximal production of 18-hydroxycorticosterone was found as 1.43 nmole/min/ mg protein and that of aldosterone 0.33 nmole/min/mg protein. The Michaelis constant for both 18-hydroxycorticosterone and aldosterone was 6.6 × 10 −6M. Q 10 (average between 20°C and 40°C) was 2.16 for the reaction corticosterone → 18-hydroxycorticosterone and 1.34 for the reaction corticosterone → aldosterone. The mitochondrial enzyme system did not 18-oxygenate exogenous 11-deoxycorticosterone, 11-dehydrocorticosterone, 20β-dihydro-corticosterone. Exogenous 18-hydroxycorticosterone and aldosterone were not metabolized. The kinetics of the transformation of corticosterone to 18-oxygenated metabolites suggested a parallel pseudo-first order reaction rather than a series pseudo-first order reaction. 18-Oxygenation of corticosterone was strongly inhibited by d,1-18-hydroxycorticosterone, p-chloromercuribenzoate, carbon monoxide, metopirone (competitive inhibition; K i = 3.0 × 10 −6 M for 18-hydroxycorticosterone and 9.0 × 10 −6M for aldosterone) and by aminopterin (noncompetitive inhibition; K i = for both metabolites 2.0 × 10 −5M). Protein synthesis inhibitors did not have any effect. Cytochrome-P450 was shown to be present in mitochondria by spectrophotometric measurements. Addition of corticosterone. 11-deoxycorticosterone and metopirone produced type II difference spectra. The mitochondrial P450 became saturated with either corticosterone or metopirone at a concentration of 21.8 nmoles/mg protein. From these studies it was concluded that the duck adrenal mitochondrial 18-oxygenating system differed from the mammalian adrenal system previously described, especially in regard of substrate specificity. 18-Hydroxycorticosterone, either endogenous or exogenous, could not serve as substrate for aldosterone production with duck adrenal mitochondria. However, under ordinary circumstances. 18-hydroxycorticosterone synthesis was always associated with aldosterone synthesis in rather fixed proportions. Circumstantial evidence suggested the role of cytochrome-P450 in 18-oxygenation. As to the mechanism of the corticosterone → 18-hydroxycorticosterone → aldosterone reaction, this sequence could not be proven. The possibility should not be discarded that atmospheric oxygen is introduced into a hitherto unknown intermediary substance, which by enzymatic action and/or chemical rearrangement gives rise simultaneously to 18-hydroxy-corticosterone and aldosterone.

Interaction of steroids with the heme protein P-450 partially purified from bovine adrenocortical mitochondria

The transformation of exogenous, isotopically labelled corticosterone to 18-hydroxycorti-costerone and aldosterone by domestic duck (Anas platyrhynchos) adrenal gland mitochondria was studied. The mitochondrial 18-oxygenating system was NADPH dependent. K+, Na+, Ca2+ and Mg2+ were necessary for maximal enzymatic activity. The enzyme present in 1 mg of mitochondrial protein became saturated with 13.2 βM of substrate (13.8 μg). Under saturation conditions the maximal production of 18-hydroxycorticosterone was found as 1.43 nmole/min/ mg protein and that of aldosterone 0.33 nmole/min/mg protein. The Michaelis constant for both 18-hydroxycorticosterone and aldosterone was 6.6 × 10−6M. Q10 (average between 20°C and 40°C) was 2.16 for the reaction corticosterone → 18-hydroxycorticosterone and 1.34 for the reaction corticosterone → aldosterone. The mitochondrial enzyme system did not 18-oxygenate exogenous 11-deoxycorticosterone, 11-dehydrocorticosterone, 20β-dihydro-corticosterone. Exogenous 18-hydroxycorticosterone and aldosterone were not metabolized. The kinetics of the transformation of corticosterone to 18-oxygenated metabolites suggested a parallel pseudo-first order reaction rather than a series pseudo-first order reaction. 18-Oxygenation of corticosterone was strongly inhibited by d,1-18-hydroxycorticosterone, p-chloromercuribenzoate, carbon monoxide, metopirone (competitive inhibition; Ki = 3.0 × 10−6 M for 18-hydroxycorticosterone and 9.0 × 10−6M for aldosterone) and by aminopterin (noncompetitive inhibition; Ki = for both metabolites 2.0 × 10−5M). Protein synthesis inhibitors did not have any effect. Cytochrome-P450 was shown to be present in mitochondria by spectrophotometric measurements. Addition of corticosterone. 11-deoxycorticosterone and metopirone produced type II difference spectra. The mitochondrial P450 became saturated with either corticosterone or metopirone at a concentration of 21.8 nmoles/mg protein. From these studies it was concluded that the duck adrenal mitochondrial 18-oxygenating system differed from the mammalian adrenal system previously described, especially in regard of substrate specificity. 18-Hydroxycorticosterone, either endogenous or exogenous, could not serve as substrate for aldosterone production with duck adrenal mitochondria. However, under ordinary circumstances. 18-hydroxycorticosterone synthesis was always associated with aldosterone synthesis in rather fixed proportions. Circumstantial evidence suggested the role of cytochrome-P450 in 18-oxygenation. As to the mechanism of the corticosterone → 18-hydroxycorticosterone → aldosterone reaction, this sequence could not be proven. The possibility should not be discarded that atmospheric oxygen is introduced into a hitherto unknown intermediary substance, which by enzymatic action and/or chemical rearrangement gives rise simultaneously to 18-hydroxy-corticosterone and aldosterone.