Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsome s by using three successive DEAE-cellulose, and Sephadex G-100 column chrom atographies. It was purified 54-fold and the yield was 23.5% with respect t o microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 /- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b 5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep li ver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K-m and V-max values were calculated to be 0.088 mu M cytochrome b5 and 315.8 mu M cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cyt ochrome b5 by reductase was studied and K-m and V-max values were determine d to be 5 mu M cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enz yme, respectively. The K-m and V-max values for the cofactor NADH in the pr esence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. N ADH-cytochrome b5 reductase was also partially purified from the same sourc e, detergent solubilized sheep liver microsomes, by using two successive DE AE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was p urified 144-fold and the yield was 7% with respect to microsomes. The appar ent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE, When ferricyanide was used as an electron acceptor, reductase showed maximu m activity between 6.8 and 7.5. The K-m and V-max values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mu mol ferric yanide reduced/min/mg enzyme, respectively. The K-m and V-max values for th e cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mu mol ferricyanide reduced/min/mg enzyme , respectively. (C) 1999 Published by Elsevier Science Ltd. All rights rese rved.