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

1988 ◽  
Vol 253 (2) ◽  
pp. 549-552 ◽  
Author(s):  
H Postlind ◽  
K Wikvall
Keyword(s):  
Vitamin D3 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Hydroxylase Activity ◽  
Ferredoxin Reductase ◽  
Pig Kidney ◽  
Nadph Cytochrome ◽  
Kidney Microsomes ◽  
Cytochrome P 450

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.

scholarly journals 25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria

1988 ◽  
Vol 252 (1) ◽  
pp. 207-213 ◽  
Author(s):  
H Dahlbäck ◽  
K Wikvall
Keyword(s):  
Vitamin D3 ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Liver Mitochondria ◽  
Polyacrylamide Gel ◽  
Ferredoxin Reductase ◽  
Chain Cleavage ◽  
Alpha 7 ◽  
Cytochrome P 450

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.

scholarly journals Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

1990 ◽  
Vol 270 (2) ◽  
pp. 345-350 ◽  
Author(s):  
T Bergman ◽  
H Postlind
Keyword(s):  
Vitamin D3 ◽  
Lauric Acid ◽  
Liver Microsomes ◽  
Hydroxylase Activity ◽  
Pig Liver ◽  
Pig Kidney ◽  
Sds Page ◽  
Kidney Microsomes ◽  
Alpha 7 ◽  
Cytochrome P 450

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.

scholarly journals Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin

1982 ◽  
Vol 204 (1) ◽  
pp. 103-109 ◽  
Author(s):  
J F Sinclair ◽  
P R Sinclair ◽  
J F Healey ◽  
E L Smith ◽  
H L Bonkowsky
Keyword(s):  
Chick Embryo ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cobalt Protoporphyrin ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.

One-electron reduction of mitomycin c by rat liver: Role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

Xenobiotica ◽  
1990 ◽  
Vol 20 (9) ◽  
pp. 967-978 ◽  
Author(s):  
R. M. Vromans ◽  
R. van de Straat ◽  
M. Groeneveld ◽  
N. P. E. Vermeulen
Keyword(s):  
Mitomycin C ◽  
Rat Liver ◽  
Nadph Cytochrome ◽  
Electron Reduction ◽  
Cytochrome P 450

scholarly journals Accelerated hepatic haem catabolism in the selenium-deficient rat

1977 ◽  
Vol 168 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R F Burk ◽  
M A Correia
Keyword(s):  
Urinary Excretion ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Haem Oxygenase ◽  
Cytochrome P 450

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.

THE ROLE OF NADPH-CYTOCHROME P-450 REDUCTASE AND TYPE I BINDING IN LIVER MICROSOMAL N-DEMETHYLATION OF ETHYLMORPHINE IN MICE

Abstracts ◽  
1977 ◽  
pp. 383
Author(s):  
A.P. van den Berg ◽  
J. Noordhoek ◽  
E.M. Savenije-Chapel ◽  
E. Koopman-Kool
Keyword(s):  
Type I ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Purification and properties of human placental NADPH–cytochrome P-450 reductase

1986 ◽  
Vol 64 (3) ◽  
pp. 184-193 ◽  
Author(s):  
Norio Muto ◽  
Liat Tan
Keyword(s):  
Salt Concentration ◽  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Visible Region ◽  
Relative Mass ◽  
Affinity Column ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Human placental NADPH–cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 μM 4-androstene-3, 17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35–60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2′, 5′-ADP–Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolyis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 ± 0.7 μmol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

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