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.     

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.     

Lack of regio- and stereospecificity in oxidation of (+) camphor by Streptomyces griseus enriched in cytochrome P-450soy

Oxidation of (+) camphor by cytochrome P-450soy-enriched intact cells of Streptomyces griseus resulted in the formation of one major and several minor metabolites. The minor metabolites were identified as 3-endo-hydroxycamphor (2%), 5-endo-hydroxycamphor (7%), 5-exo-hydroxycamphor (9%), 2,5-diketobornane (2%), and camphorquinone (3%). The major metabolite was isolated and conclusively identified as 6-endo-hydroxycamphor (60%). When supplemented with NADPH, spinach ferredoxin:NADP oxidoreductase and spinach ferredoxin, homogeneous preparations of cytochrome P-450soy oxidized camphor to a mixture of 3-endo-, 5-endo-, 5-exo-and 6-endo-hydroxycamphor. The data presented indicates that cytochrome P-450soy resembles its mammalian counterparts in its lack of regio- and stereospecificity in camphor oxidation.     

Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy and ferredoxinsoy (soyB) from Streptomyces griseus

Xenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P-450, designated cytochrome P-450soy. A DNA segment carrying the structural gene encoding P-450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P-450soy was compared with that for P-450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe-4S] ferredoxin (soyB), named ferredoxinsoy, was identified.     

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.     

Isolierung von Cytochrom P-450 und des entsprechenden Reductasesystems aus Acinetobacter calcoaceticus

Cytochrome P–450 is found in soluble as well as in particulate fractions of n-alkane-induced cells of Acinetobacter calcoaceticus EB 104 as shown by differential centrifugation and; gelfiltration experiments. It is reduced by NADH in the presence of at least two soluble proteins. Particulate cytochrome P–450 was extracted by Triton X-100 and purified to homogeneity. The 80 fold enrichment resulted in a content of 19.0 nmol cytochrome P-450/mg protein. The maxima of the absolute spectra are 417 nm for the oxidized, 408 nm for the reduced and 448 nm for the CO complex of reduced cytochrome. A ferredoxin, identified by maxima of 415 and 460 nm in the absorption spectrum of the oxidized form and by the occurrence of acid-labile sulfur, as well as a corresponding NADH-dependent ferredoxin reductase were isolated independently from the same bacterial strain. Enzymatic reduction of purified cytochrome P–450 is mediated by these proteins, which are discussed as in vivo components of the cytochrome P–450 system of A. calcoaceticus EB 104.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

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.     

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.     

Purification of a Single Major Form of Microsomal Cytochrome P-450 from Tulip Bulbs (Tulipa gesneriana L.)

Microsomal cytochrome P-450 from tulip bulbs (Tulipa gesneriana L., Balalaika) was purified to an almost electrophoretically homogeneous preparation. The specific content of cytochrome P-450 in the final preparation was 6.68 nmol/mg protein, which was 30-fold enriched from that of the solubilized fractions of microsomes. The molecular weight of purified cytochrome P-450 by SDS-gel electrophoresis is 52,500. The Oxidized form of the purified cytochrome P-450 had absorption peaks at 392, 552, and 645 nm and the absolute reduced CO spectrum peaked at 448 nm. Judged spectrally, the purified cytochrome P-450 is in high spin in the oxidized state. Antiserum against this cytochrome P-450 previously has shown to be highly specific for its antigen but showed a single precipitin line with solubilized microsomal proteins from tulip bulbs of several other cultivars. The physiological role of this cytochrome P-450, however, is unknown in these dormant tulip bulbs.     

Evidence for the formation of 26-hydroxycholesterol by cytochrome P-450 in pig kidney mitochondria

Pig kidney mitochondria were found to catalyze the formation of 26-hydroxycholesterol, an inhibitor of cholesterol biosynthesis. The cholesterol 26-hydroxylase was purified 600-fold. It was present in a mitochondrial enzyme fraction enriched in cytochrome P-450. The cytochrome P-450 fraction required NADPH, mitochondrial ferredoxin and ferredoxin reductase for 26-hydroxylase activity. The mitochondria and the purified 26-hydroxylase preparation also catalyzed 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and intermediate in cholic acid biosynthesis, and of 25-hydroxyvitamin D3. The role of extra-hepatic formation of 26-hydroxycholesterol is discussed.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

Cytochrome P-450 from lodderomyces elongisporus: Its purification and some properties of the highly purified protein

An effective method, based on the chromatography on ω-aminooctyl Sepharose 4B, for the purification of the alkane-induced cytochrome P-450 is described. The purified cytochrome P-450 was homogeneous in SDS/polyacrylamide gel electrophoresis. In the oxidized state it showed a low spin type absorption spectrum. The reduced CO-complex is characterized by a Soret peak at 447 nm. The alkane hydroxylating enzyme system could be reconstituted combining purified cytochrome P-450 with partially purified NADPH-cytochrome P-450 reductase from the yeast microsomal fraction.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Characterization and identification of a pyrazole-inducible form of cytochrome P-450

In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.     

Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17alpha-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.