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 Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1)

1989 ◽  
Vol 260 (1) ◽  
pp. 81-85 ◽  
Author(s):  
D J Waxman ◽  
D P Lapenson ◽  
J J Morrissey ◽  
S S Park ◽  
H V Gelboin ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Product ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Liver Microsomes ◽  
Parental Cell Line ◽  
Rat Liver Microsomes ◽  
Hydroxylase Activity ◽  
Specific Content ◽  
Cytochrome P 450

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.

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

Interlaboratory comparison of total cytochrome P-450 and protein determinations in rat liver microsomes

1987 ◽  
Vol 61 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Alphons A. J. J. L. Rutten ◽  
Hein E. Falke ◽  
Jan F. Catsburg ◽  
Randy Topp ◽  
Bas J. Blaauboer ◽  
...  
Keyword(s):  
Rat Liver ◽  
Interlaboratory Comparison ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Cytochrome P 450

Pituitary regulation of cytochrome P-450-mediated metabolism of steroids and xenobiotics in rat liver microsomes

1986 ◽  
Vol 7 (4) ◽  
pp. 575-582 ◽  
Author(s):  
Agneta Blanck ◽  
Anders Åström ◽  
Tiiu Hansson ◽  
Joseph W. De Pierre ◽  
Jan-Åke Gustafsson
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Cytochrome P 450
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