Purification and characterization of two forms of microsomal cytochrome b5 from sheep lung.
Two forms of cytochrome b5 were purified from detergent solubilized sheep lung microsomes by three successive DEAE-cellulose, Sephadex G-100 and Sephadex G-200 column chromatographies. The specific contents of cytochromes b5-I and b5-II were determined to be 45.4 and 43.8 nmol b5/mg protein, which represented up to 567 and 547-fold purification compared with that of the lung microsomes. The most striking difference between b5-I and b5-II was observed in their elution pattern from the third DEAE-cellulose column. Cytochromes gave one protein band with a Mr of 16400 +/- 500 on SDS-PAGE. Both forms of reduced b5 showed a major peak at 423 nm while reduced b5-I had two minor peaks at 527 and 556 and reduced b5-II gave two well-defined peaks at 526 and 555 nm. The ability of the purified b5-I and b5-II fractions to transfer the electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. Apparent Km, 0.055 microM, of b5-II was found to be 38% lower than that of b5-I. In addition, cytochrome b5-I was found to be more sensitive to heat treatment than b5-II when cytochromes were subjected to 62 degrees C for varying periods of time and the coupling of b5 reduction to cytochrome c reduction was determined. These results may indicate that two forms may exist in lung endoplasmic reticulum.
Başaran
N
N
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
Arinç
E
E
D000818:Animals; D015786:Cytochromes b5 / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D012756:Sheep
D015786:Cytochromes b5