Immunopurified 25-hydroxyvitamin D 1 alpha-hydroxylase and 1,25-dihydroxyvitamin D 24-hydroxylase are closely related but distinct enzymes.
The chick kidney mitochondrial cytochrome P-450 1,25-dihydroxyvitamin D3 24-hydroxylase was partially purified by sequential polyethylene glycol precipitation, aminohexyl-Sepharose 4B, and hydroxylapatite chromatography. The specific activity of the final preparation, when reconstituted with NADPH, adrenodoxin, and adrenodoxin reductase, was 245 pmol/min/mg of protein or 0.56 pmol/min/pmol of P-450. The specific cytochrome P-450 content was 0.45-0.73 nmol/mg of protein. BALB/c mice immunized with this preparation developed serum polyclonal antibodies to the 24-hydroxylase, as demonstrated by immunoprecipitation. Splenic lymphocytes from an immunized mouse were fused with myeloma NSI/1-Ag-4-1 cells, and hybridomas secreting monoclonal antibodies to the 24-hydroxylase were detected by immunoprecipitation. The hybridoma lines were cloned by limiting dilution and further characterized as IgG1, IgG3, and IgM subclasses. In one-dimensional immunoblots of soluble 24-hydroxylase preparations, the monoclonal antibodies revealed a single band with an apparent molecular weight of 59,000. The monoclonal antibodies did not cross-react with cytochrome P-450s from other species but immunoprecipitated and immunoblotted a soluble chick renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase preparation, demonstrating the close similarity of these two hydroxylases. These antibodies were coupled to Sepharose CL-4B and used to isolate to homogeneity the two enzymes from chick kidney mitochondria. Amino-terminal sequences and amino acid composition data demonstrate that these enzymes are different but homologous.
Burgos-Trinidad
M
M
Department of Biochemistry, University of Wisconsin-Madison, College of Agricultural and Life Sciences 53706.
Ismail
R
R
Ettinger
R A
RA
Prahl
J M
JM
DeLuca
H F
HF
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002478:Cells, Cultured; D002645:Chickens; D002845:Chromatography / Q000379:methods; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D006882:Hydroxyapatites; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000302:isolation & purification* / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D000911:Antibodies, Monoclonal; D006882:Hydroxyapatites; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase