Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome.
A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D002417:Cattle; D003573:Cytochrome b Group / Q000097:blood* / Q000302:isolation & purification; D003902:Detergents; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D006098:Granulocytes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D019255:NADPH Oxidases; D017830:Octoxynol; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D011485:Protein Binding; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003902:Detergents; D002245:Carbon Dioxide; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C036888:cytochrome b558; D019255:NADPH Oxidases; D008058:Dihydrolipoamide Dehydrogenase