title: Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases from vitamin D-replete pigs.
abstract: Pig kidney mitochondrial 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities have been solubilized with cholate/Emulgen 911 and reconstituted with NADPH, ferredoxin reductase and ferredoxin. All three of these components are required for full catalytic activity of both enzymes. Both products were identified by co-chromatography with authentic metabolites on both normal and reverse-phase h.p.l.c. using solvent systems which were shown to separate 10-oxo-19-nor-25-hydroxyvitamin D3 from 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3]. In addition, periodate treatment of the 24,25-(OH)2-D3 product resulted in complete loss of the product as measured by protein-binding assay. Further purification by p-chloroamphetamine-Sepharose chromatography of a solubilized extract from a pig fed a normal diet increased the specific content of the cytochrome P-450 from 0.019 to 0.239 nmol/mg and the 1 alpha-hydroxylase activity from 4.75 to 268 pmol/h per mg. Activity of the 24-hydroxylase in the crude solubilized extract was 6.3 pmol/h per mg, but was undetectable after partial purification by a p-chloroamphetamine-Sepharose column. However, further fractionation of this material by DEAE-Sepharose chromatography resulted in a further increase in 1 alpha-hydroxylase activity to 430 pmol/h per mg and detection of 24-hydroxylase in a separate fraction at a level of 53 pmol/h per mg. Production of 1,25-(OH)2-D3 was linear with time up to 2 h and was dependent upon ferredoxin concentration as well as cytochrome P-450 concentration in the range of 0-40 nM. In the presence of excess ferredoxin and adequate amounts of cytochrome P-450, 1,25-(OH)2-D3 production was also dependent upon substrate concentrations in the range of 0.25 to 2.5 microM yielding an estimated Km of 1 microM. In the presence of excess substrate and ferredoxin, the catalytic-centre activity of the enzyme was estimated to be 1 h-1.
authors.affiliation: Department of Medicine, Medical College of Wisconsin, Milwaukee 53226.
authors.forename: R W
authors.initials: RW
authors.lastname: Gray
authors.forename: J G
authors.initials: JG
authors.lastname: Ghazarian
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004032:Diet; D007672:Kidney Cortex / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D012995:Solubility; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine; D014807:Vitamin D / Q000008:administration & dosage; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase