Isolation of chick renal mitochondrial ferredoxin active in the 25-hydroxyvitamin D3-1alpha-hydroxylase system.
An iron-sulfur protein has been isolated from chick kidney mitochondria and purified (200-fold as determined enzymatically by its NADPH-cytochrome c reductase activity in the presence of adrenodoxin reductase) on DEAE-cellulose and gel filtration on Sephadex G-100. The purified protein showed an absorption peak at 411 nm with a shoulder at 460 nm. The electron paramagnetic resonance spectrum was typical of a ferredoxin-type iron-sulfur protein with g values: gx=gy-1.94 and gz=2.02. The molecular weight was estimated by gel filtration to be 12,500. When tested against anti-adrenodoxin gamma-globulin, the protein showed a precipitin line that fused completely with that of adrenodoxin. Based on these findings it is concluded that this protein is an iron-sulfur protein quite similar to adrenal ferredoxin. In the presence of adrenoxodin reductase, NADPH, and carbon monoxide, the purified renal ferredoxin was found to be active in the reduction of cytochrome P-450 solubilized from chick kidney mitochondria. It was also effective in the reconstituted 25-hydroxyvitamin D3-1alpha-hydroxylase composed of the cytochrome P-450 from rachitic chick kidneys and adrenodoxin reductase. A ferredoxin reductase isolated from chick kidney mitochondria could replace adrenodoxin reductase in the reconstituted system. These results strongly support a previous conclusion that the kidney mitochondrial 25-hydroxyvitamin D3-1alpha-hydroxylation system consists of a renal ferredoxin reductase (presumably a flavoprotein), renal ferredoxin, and cytochrome P-450.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D000311:Adrenal Glands / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002645:Chickens; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000378:metabolism*
D005288:Ferredoxins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase