Immunopurified 25-hydroxyvitamin D 1 alpha-hydroxylase and 1,25-dihydroxyvitamin D 24-hydroxylase are closely related but distinct enzymes.

摘要: 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. specific activity of the final preparation, when reconstituted with NADPH, adrenodoxin, adrenodoxin reductase, 245 pmol/min/mg protein or 0.56 pmol/min/pmol P-450. content 0.45-0.73 nmol/mg protein. BALB/c mice immunized this preparation developed serum polyclonal antibodies to 24-hydroxylase, as demonstrated immunoprecipitation. Splenic lymphocytes from an mouse were fused myeloma NSI/1-Ag-4-1 cells, hybridomas secreting monoclonal detected hybridoma lines cloned limiting dilution further characterized IgG1, IgG3, IgM subclasses. In one-dimensional immunoblots soluble preparations, revealed a single band apparent molecular weight 59,000. did not cross-react P-450s other species but immunoprecipitated immunoblotted renal 25-hydroxyvitamin 1 alpha-hydroxylase demonstrating close similarity these two hydroxylases. These coupled Sepharose CL-4B used isolate homogeneity enzymes mitochondria. Amino-terminal sequences amino acid composition data demonstrate that are different homologous.