Purification and biochemical characterization of reno-ferredoxin from bovine kidney mitochondria.

Bovine reno-ferredoxin was purified from kidney mitochondria by an improved method that included hydrophobic and ion-exchange chromatography on Toyopearl gels. The optical absorption spectrum of the oxidized reno-ferredoxin revealed two peaks, at 414 and 455 nm in the visible region. The minimum molecular weight of the ferredoxin was 12,900 Da by SDS-polyacrylamide gel electrophoresis. The amino acid residues of the NH2-terminal sequence of the ferredoxin were investigated using by a gas-phase sequencer. Bovine reno-ferredoxin and adreno-ferredoxin showed almost identical NH2-terminal amino acid sequences. Reconstitution of the 25-hydroxyvitamin D3-1 alpha -hydroxylase system was performed with the following three components: NADPH-ferredoxin reductase from bovine kidney mitochondria, reno-ferredoxin, and cytochrome P-450(D1 alpha) from bovine kidney mitochondria. The results demonstrated that the reno-ferredoxin was essential for the 1 alpha-hydroxylase activity of 25-hydroxyvitamin D3.