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 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 Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes

1992 ◽  
Vol 287 (3) ◽  
pp. 725-731 ◽  
Author(s):  
E Axén ◽  
T Bergman ◽  
K Wikvall
Keyword(s):  
Liver Enzyme ◽  
Rat Liver ◽  
Vitamin D3 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Pig Liver ◽  
Specific Content ◽  
Apparent Homogeneity ◽  
Structural And Functional Properties ◽  
Cytochrome P 450

A cytochrome P-450 which catalyses 25-hydroxylation of vitamin D3 has been purified to apparent homogeneity from pig liver microsomes. The specific content of cytochrome P-450 was 12 nmol.mg of protein-1, and the preparation showed a single band with an apparent M(r) of 50,500 upon SDS/PAGE. A monoclonal antibody raised against the vitamin D3 25-hydroxylase reacted strongly with the purified 25-hydroxylating cytochrome P-450 from pig kidney microsomes [Bergman & Postlind (1990) Biochem. J. 270, 345-350]. The liver enzyme showed structural and functional properties very similar to those of the kidney enzyme. The two enzymes differed with respect to only one of the first 16 N-terminal amino acids. The vitamin D3 25-hydroxylase in pig liver microsomes exhibited a turnover and an apparent Km for 25-hydroxylation of vitamin D3 which were of the same order of magnitude as those of a well-characterized male-specific 25-hydroxylating cytochrome P-450 in rat liver microsomes. The two enzymes differed structurally. The pig liver enzyme was, in contrast to the rat liver enzyme, not sex-specific, and did not catalyse 16 alpha-hydroxylation of testosterone. These properties of the 25-hydroxylase in rat liver microsomes have led to questions on the role of microsomal 25-hydroxylation of vitamin D3. It is concluded that studies on microsomal 25-hydroxylation with the rat may be misleading. The results of the present study show that the pig appears to be a representative species for evaluation of vitamin D3 hydroxylases in other mammals, including man.

scholarly journals Characterization of mitochondrial cytochromes P-450 from pig kidney and liver catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids

1991 ◽  
Vol 276 (2) ◽  
pp. 427-432 ◽  
Author(s):  
T Bergman ◽  
H Postlind
Keyword(s):  
Monoclonal Antibody ◽  
Protein Band ◽  
Similar Species ◽  
Mitochondrial Cytochrome ◽  
Pig Liver ◽  
Kidney Mitochondria ◽  
Pig Kidney ◽  
Sds Page ◽  
Liver And Kidney ◽  
Cytochrome P 450

The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.

scholarly journals The omega- and (omega-1)-hydroxylase activities of prostaglandins A1 and E1 and lauric acid by pig kidney microsomes and a purified kidney cytochrome P-450.

1981 ◽  
Vol 256 (12) ◽  
pp. 5961-5964 ◽  
Author(s):  
R.T. Okita ◽  
L.K. Parkhill ◽  
Y. Yasukochi ◽  
B.S. Masters ◽  
A.D. Theoharides ◽  
...  
Keyword(s):  
Lauric Acid ◽  
Pig Kidney ◽  
Kidney Microsomes ◽  
Cytochrome P 450

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

1986 ◽  
Vol 250 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Myrtle Thierry-Palmer ◽  
Suzanne Cullins ◽  
Shakoora Rashada ◽  
T.Kenney Gray ◽  
Almena Free
Keyword(s):  
Rat Liver ◽  
Vitamin D3 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Hydroxylase Activity
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