Purification of cytochrome P-450 from bovine adrenocortical mitochondria by an “Aniline-sepharose” and the properties

doi:10.1016/S0006-291X(75)80424-4

Biochemical and Biophysical Research Communications

Volume 63, Issue 3, 7 April 1975, Pages 588-593

https://doi.org/10.1016/S0006-291X(75)80424-4 Get rights and content

Summary

Cytochrome P-450 has been purified by an affinity chromatography using an aniline substituted Sepharose column. The preparation is essentially homogeneous as judged by electrophoresis and ultracentrifugation. The absorbance ratio(A394nm/A280nm) is 0.83 and the protein weight per heme is 80 kg. As determined by the sedimentation equilibrium method, the molecular weight is 200 kilodaltons in native protein and 46 kilodaltons in guanidine-treated protein, respectively. The preparation in oxidized state has a high spin type absorption spectrum. When the preparation is treated with the adrenal ferredoxin-dependent electron transfer system, the spectrum is converted into that of a low spin type which is reconverted into the high spin form by the addition of cholesterol.

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On SDS–polyacrylamide gel electrophoresis, only one protein band for each purified preparation was observed, indicating that the two P450 preparations had been satisfactorily separated in purified forms for further characterization. When the preparations were carboxymethylated in the presence of 6 M guanidine HCl and 100 mM 2-mercaptoethanol, the sedimentation equilibrium data gave molecular weights of 46,000 and 43,000 for P450scc and P45011β, respectively [25,26]. Under a newly developed sensitive assay system, the purified P450scc preparation specifically catalyzed the side-chain cleavage of cholesterol to form pregnenolone with a turnover number of 16 min−1 in the presence of adrenodoxin and NADPH-adrenodoxin reductase, but did not catalyze 11β- or 18-hydroxylase activity for deoxycorticosterone.
A simple and rapid method to measure cholesterol binding to P450s and other proteins
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Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. Using three cholesterol-metabolizing cytochrome P450s (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar to micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flow-through fraction, whereas P450 binds to the membrane. The radioactivity of the membranes is then measured, and a saturation curve is generated after correction for nonspecific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate the interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another binding negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions.
Thus, the cholesterol binding properties of a wide variety of proteins could be characterized using this filter assay.
Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450<inf>tu</inf>) from mouse liver tumors
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Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15α-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450_tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent M_r of 50000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450_tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15α-hydroxylation of progesterone (2.3 min⁻¹) and testosterone (2.5 min⁻¹), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450_15α, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450_tu differed from P-450_15α by its higher molecular weight, its 40-times lower steroid 15α-hydroxylation and its 4-times higher benzphetamine N-demethylation.
Existence of multiple forms of cytochrome P-450<inf>scc</inf> purified from bovine adrenocortical mitochondria
1987, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Three fractions of cytochrome P-450_scc (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-450_scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450_scc 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 t he sequence of fraction c showed a missing of isoleucine at the NH₂-terminal. COOH-terminal amino-acid sequences of fraction 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-450_scc, 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-450_scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450_scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.
Structure of genes encoding steroidogenic enzymes
1987, Journal of Steroid Biochemistry
Synthesis of adrenal steroid hormones from cholesterol entails the actions of only five enzymes, four of which are specific forms of cytochrome P450. These cytochrome P450 enzymes have all been isolated and their activities reconstituted in vitro, showing that each enzyme catalyses multiple steroidal conversions. Genes or complementary DNAs have been cloned for human P450scc (the cholesterol side-chain cleavage enzyme), P450cl7 (17α-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase). The sequences for microsomal P450cl7 and P450c21 are much more closely related to one another than either is to the sequence for mitochondrial P450scc. Each of these P450 enzymes is encoded by a single human gene; the gene for P450scc lies on chromosome 15, that for P450c 17 lies on chromosome 10, and that for P450c21 lies on chromosome 6. The human, mouse and bovine genomes each have two P450c21 genes. While only one of these is active in mouse and man, both genes may he active in cattle. A wide variety of lesions in the human P450c21 (B) gene causes congenital adrenal hyperplasia, a common genetic disorder.
Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria
1986, FEBS Letters
Hepatic ferredoxin (hepatoredoxin) was purified from bovine liver mitochondria. The monomeric molecular mass of the hepatoredoxin was larger than that of adrenocortical ferredoxin (adrenodoxin) from bovine adrenocortical mitochondria at 14 kDa. We studied the amino acid residues and NH₂-terminal sequence of this protein. The hepatoredoxin was organ-specific protein. The optical absorption spectrum of oxidized hepatoredoxin had two peaks, at 414 and 455 nm in the visible region. Hepatoredoxin formed an immunoprecipitin lime against anti-adrenodoxin immunoglobulin in Ouchterlony double diffusion, and an immunochemical staining band in Western blotting.

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^*: A part of the results was presented at the meeting of International Congress of Biochemistry in Stockholm 1973(1). This work was supported by the Scientific Research Fund B-947051 from the Ministry of Education of Japan.

^†: Present addresses: Central Research Institute, Suntory Ltd., Osaka 618.

^§: Present addresses: Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Osaka 532.

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Purification of cytochrome P-450 from bovine adrenocortical mitochondria by an “Aniline-sepharose” and the properties*

Summary

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Methods in Enzymology