Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1α-hydroxylase

Abstract

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.

Reciprocal post-translational regulation of renal 1α- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin. mRNA-directed cell-free synthesis and immunoisolation of ferredoxin

“…Nuclease-treated rabbit reticulocyte lysate was purchased from Promega Biotech and prestained molecular-mass markers were from Integrated Separation Systems. Pansorbin cells and the detergent Nonidet P40 were obtained from Calbiochem; Autofluor autoradiography enhancer was from National Diagnostics; Kodak XAR-5 films were from Winchester X-Ray Inc. Monospecific anti-ferredoxin was obtained as reported previously [13].…”

Mixed-function oxidases of 25-hydroxycholecalciferol in isolated chick kidney glomeruli: Evidence for nuclear localization

“…However, we could not exclude the possibility that the intense glomerular fluorescence was not a result of a reaction between the antibodies and a ferredoxin-type protein present in glomeruli which possesses biochemical functions unrelated to bioactivation of 25-OH-D3. The immunogenic similarity of beef adrenal ferredoxin (steroidogenic) to the chick renal ferredoxin despite the differences in their biochemical functions is a clear example [8]. Hence, it was essential to demonstrate that the glomerular immunofluorescence could be correlated .…”

“…Recent studies utilizing immunofluorescent techniques [6] have shown that monospecific antibodies raised against the purified ferredoxin component of the la-hydroxylase [8] cross-react with kidney antigens predominantly localized in glomerular and proximal tubular segments of the nephron. The immunogenic reactivity in the tubular segment corroborates other reports concerning the localization of the lo~-hydroxylase in this portion of the nephron [9].…”

“…The properties of the two hydroxylases are well defined [4,5]. Recently, using indirect immunofluorescent technique, we demonstrated [6] that the ferredoxin component of the hydroxylases [7,8] is predominantly localized in the glomeruli and proximal convoluted tubules of chick kidney nephrons. This localization in the proximal tubule was consistent with other reports [9,10].…”