Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1 alpha-hydroxylase.
The chick kidney mitochondrial iron--sulphur protein (ferredoxin), a component of the NADPH--cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH--cytochrome c reductase assay utilizing crude renal NADPH--ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000302:isolation & purification; D005779:Immunodiffusion; D007668:Kidney / Q000032:analysis; D008928:Mitochondria / Q000032:analysis; D013250:Steroid Hydroxylases
D000596:Amino Acids; D005288:Ferredoxins; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase