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.     

Crystallization of cytochrome P-450scc from bovine adrenocortical mitochondria

Cytochrome P-450scc (P-450scc), a cholesterol side-chain cleavage enzyme from bovine adrenocortical mitochondria, has been crystallized for the first time. Upon removal of glycerol from the solution of the native enzyme complexed with pyridoxal 5'-phosphate (PLP) by microdialysis against distilled water, reddish and planar crystals appeared. The crystals of native P-450scc were also obtained by the same procedure. We identified the crystals as the P-450scc-PLP complex or native P-450scc by absorption spectroscopy and SDS-polyacrylamide gel electrophoresis, and characterized them under a polarization microscope.     

The active form of cytochrome P-450 from bovine adrenocortical mitochondria.

Cytochrome P-450 from bovine adrenocortical mitochondria exists in three forms of molecular weight: 850,000 (protein 16), of one-half (protein 8), and of one-quarter of this value (protein 4). The forms of the enzyme are named according to the number of subunits and all appear to be active in converting cholesterol to 3beta-hydroxy-5-pregnen-20-one (side chain cleavage) (Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5606). To determine whether all three forms are active at their characteristic molecular weights, the three cytochromes were each layered onto separate sucrose density gradients and centrifuged at 49,000 rpm for 60 min; the gradients contained all the factors necessary for side chain cleavage including one of the following substrates: cholesterol, 20S-hydroxycholesterol, and 20S,22R-dihydroxycholesterol. Regardless of the form of P-450 layered onto the gradient and regardless of the substrate, enzyme activity (side chain cleavage) was observed only in fractions corresponding to a sedimentation coefficient of 20 to 22 S which is that for protein 16. No activity was observed at S values corresponding to either protein 8 or protein 4. These findings indicate that the active form of cytochrome P-450 from adrenocortical mitochondria is that containing 16 subunits, i.e. the form in which the cytochrome is normally isolated from adrenal mitochondria. Forms consisting of eight and four subunits which can be prepared from protein 16 become active only by forming protein 16, at least in an aqueous medium in vitro.     

Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria - inhibitory effect of puromycin.

$\text{Trans}$-3-dehydro-D, L-ornithine and $\text{trans}$-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of ornithine decarboxylase. The K_I′S for $\text{trans}$-3-dehydro-L-ornithine and $\text{trans}$-1, 4-diamino-2-butene are 2.2 and 2.0 μM respectively. Both analogs bind much more tightly to the enzyme than either ornithine or putrescine. Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of $\text{trans}$-3-dehydro-D, L-ornithine to the culture medium.     

Cytochrome P450 in adrenocortical mitochondria

Summary Cytochrome P₄₅₀ in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P₄₅₀ each catalyzing a specified reaction have been purified to a homogeneous state, that is, P_450scc having cholesterol side chain cleavage activity and P₄₅₀₁₁ having 11-hydroxylation activity. The properties of these purified P₄₅₀'s as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P₄₅₀ in the mitochondria) preparation in situ.Among many findings, both purified cytochromes P₄₅₀ were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P₄₅₀. These effects are important from the point of view that the primary role of the heme of cytochrome P₄₅₀ is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P₄₅₀ in the reconstituted system have also been described Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.     

Cytochrome P-450-linked monooxygenase systems of bovine brain mitochondria and microsomes

1. Cytochrome P-450-linked monooxygenase of mitochondria and microsomes of the cerebrum, cerebellum and pituitary gland of bovine brain were investigated biochemically and immunochemically. 2. The cytochromes P-450scc and P-450(11) beta, NADPH-ferredoxin reductase and adreno-ferredoxin were detected and their enzymatic activities of steroid hydroxylations were found in the bovine brain.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Electron paramagnetic resonance studies of the purified cytochrome P-450scc and P-45011beta from bovine adrenocortical mitochondria.

Electron paramagnetic resonance studies have been carried out on two species of cytochrome P-450 (P-450scc and P-45011beta) purified from bovine adrenocortical mitochondria. The g values of the steroid-bound cytochromes in the high spin form were determined at 4.2 degrees K to be 8.07, 3.60 and 1.70 for P-450scc and 8.00, 3.65 and 1.71 for P-45011beta. The E/D values were estimated to be 0.103 for P-450scc and 0.099 for P-45011beta. Either high spin P-450 was converted into the low spin form by the treatment with an NADPH dependent electron donating system and subsequent gel filtration in order to remove the steroid. The g values of the low spin ferric cytochromes were 2.423, 2.247 and 1.914 for P-450scc and 2.430, 2.251 and 1.919 for P-45011beta at 77 degrees K. The values for magnitude of delta/gamma, magnitude of V/gamma and k were 5.69, 5.21 and 1.11 for P-450scc and 5.94, 5.38 and 1.16 for P-45011beta. These studies indicate that there are some differences in the ferric heme environment between P-450scc and P-45011beta.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

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

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

The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria

A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

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.