Atsuo Hiwatashi

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.

Heterogeneity of adrenocortical ferredoxin

Bovine adrenocortical ferredoxin (adreno‐ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK‐gel phenyl‐5PW column with a high‐pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH‐cytochrome c reducing system. Their amino‐terminal sequences all corresponded to the reported sequence, but the carboxyl‐terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno‐ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno‐ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.

Isolation and purification of mature bovine adrenocortical ferredoxin with an elongated carboxyl end

Mature bovine adrenocortical ferredoxin (adreno-ferredoxin) was extracted from fresh adrenal glands at pH 9.0. Extraction and purification at this alkaline pH protected the mature adreno-ferredoxin molecule from proteolytic degradation. The mature adreno-ferredoxin was extensively purified by a rapid procedure including two kinds of column chromatography, hydrophobic and ion exchange. The purified adreno-ferredoxin was homogeneous on the basis of two HPLC analyses, hydrophobic and ion exchange, and had the highest purity so far reported. Then it was digested by trypsin and the carboxyl-terminal peptide was isolated from the tryptic digest by a novel column chromatographic method using a cation-exchange HPLC column, TSK-gel SP-5PW. The carboxyl-terminal amino acid was isoleucine, so the adreno-ferredoxin had 127 amino acid residues, the longest polypeptide so far determined chemically for bovine adreno-ferredoxin. Only Glu-128 was lacking within the carboxyl-terminal elongated peptide that was found by nucleotide sequencing of the adreno-ferredoxin gene. There was no evidence obtained on whether the deletion of Glu-128 was due to so-called carboxyl-terminal processing or to proteolytic degradation during storage and purification.

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.

Purification and organ-specific properties of cholecalciferol 25-hydroxylase system: Cytochrome P-450D25-linked mixed function oxidase system

Summary A cholecalciferol 25-hydroxylase(vitamin D3 25-hydroxylase) system was purified from beef liver microsomes by affinity chromatography of sepharose with a ligand of vitamin D3. The hydroxylase system was composed of two components of NADPH-cytochrome P-450 reductase and cytochrome P-450D25. The molecular weight of cytochrome P-450D25, a glycoprotein, was 48,000. The molecular weight of NADPH-cytochrome P-450 reductase was 79,000, a value which differed slightly from that of the beef adrenocortical microsomes. The optical and physicochemical properties of cytochrome P-450D25 and NADPH-cytochrome P-450 reductase were investigated. The reconstituted vitamin D3 25-hydroxylase system was not accelerated by the NADH-dependent electron transport system of NADH-cytochrome b5 reductase and cytochrome b5. The NADPH-cytochrome P-450 reductase as well as cytochrome P-450D25 were chemically and immunochemically different from the steroid 21-hydroxylase system of beef adrenocortical microsomes.

Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase of pig adrenocortical mitochondria.

NADPH-adrenodoxin reductase is a component of an adrenodoxin-linked cytochrome P-450 mixedfunction oxidase system of adrenocortical mitochondria, and many steroids are hydroxylated by this mixed-function oxidase system [l] . We have reported previously on crystalline NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria [2,3] . This communication describes the crystallization of pig NADPH-adrenodoxin reductase from pig adrenocortical mitochondria by two methods and the properties of the NADPH-adrenodoxin reductase.

The role of the sugar regions of components of the cytochrome P-450-linked mixed-function oxidase (monooxygenase) system of bovine adrenocortical mitochondria.

Cytochrome P-450scc from bovine adrenocortical mitochondria was found to be a glycoprotein, similar to NADPH-adrenodoxin reductase. The role of sugar moieties of components of the cytochrome P-450-linked mixed-function oxidase system was investigated. The NADPH-ferricyanide reductase activity of NADPH-adrenodoxin reductase was not affected by neuraminidase treatment, but NADPH-cytochrome c reductase activity of NADPH-adrenodoxin reductase and adrenodoxin was lost. Cytochrome P-450scc treated with neuraminidase could not be reduced by NADPH-adrenodoxin reductase and adrenodoxin, and the side-chain cleavage activity of cholesterol by the cytochrome P-450-linked mixed-function oxidase system was lost by neuraminidase treatment of cytochrome P-450scc or NADPH-adrenodoxin reductase or both. These results indicate that the sugar moieties of the components of the cytochrome P-450-linked mixed-function oxidase system are necessary for binding and attaching of the components and electron transport.

Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria

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.

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.

Tissue and subcellular distributions of cholecalciferol 25-hydroxylase: Cytochrome P-450D25-linked monooxygenase system

Cholecalciferol (vitamin D3) 25-hydroxylase activity and its tissue and subcellular distributions were studied using rabbit, rat, bovine, chick, duck, guinea-pig and yellowtail. Vitamin D3 25-hydroxylase was found not only rabbit and bovine livers, kidneys and intestines, but also in lungs, pituitary glands, adrenocortices, ovaries, adipose tissues, blood vessels, brains and testes. Subcellular fractions of rabbit livers were examined for the presence of this activity and it was detected in the nuclear membranes, smooth and rough endoplasmic reticula, Golgi membranes and mitochondrial membranes. The activity in microsomes was induced by sodium phenobarbital, but not by beta-naphthoflavone and 3-methylcholanthrene, and was inhibited by isocyanide and metyrapone as inhibitor of cytochrome P-450.