Purification and characterization of microsomal cytochrome b560ms from a unicellular eukaryote Tetrahymena pyriformis.
Effective resolution of detergent-solubilized Tetrahymena microsomal cytochrome b560ms, which is similar to mammalian cytochrome b5, was obtained by chromatography on DEAE-cellulose and gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of Tetrahymena cytochrome b560ms is 22,000. The absorption peaks are 414 nm in the oxidized form and 560, 528, and 425 nm in the dithionite-reduced form. The alpha-peak of the reduced form, which is situated at 560 nm and asymmetric with a shoulder at 556 nm, is different from that of mammalian liver microsomal cytochrome b5. The spectra of the reduced and the oxidized cytochrome b560ms are not altered by the presence of cyanide, azide, and carbon monoxide. The alpha-peak is resolved into two distinct peaks at 551 and 558 nm at low temperature. The absorption peaks at 557, 527, and 418 nm in the pyridine ferrohemochrome spectrum indicate that protoheme is the prosthetic group of the purified b-type cytochrome. The oxidation-reduction potential (E'0) is -42 mV. It is reducible by NADH in the presence of an NADH-cytochrome b5 reductase purified from rat liver microsomes. The present results suggest that the pathways of electrons to microsomal cytochrome b560ms-linked oxidative desaturation of fatty acyl-CoA in protozoan Tetrahymena pyriformis are via either of the two microsomal flavoprotein reductases, NADH-ferricyanide reductase or NADPH-cytochrome c reductase.
D000818:Animals; D002248:Carbon Monoxide; D003573:Cytochrome b Group / Q000302:isolation & purification* / Q000378:metabolism; D042963:Electron Transport Complex II; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013769:Tetrahymena pyriformis / Q000378:metabolism*
D003573:Cytochrome b Group; C025355:cytochrome b560; D002248:Carbon Monoxide; D042963:Electron Transport Complex II