The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes

Abstract

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91 000 (glycosylated) and 22 000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm 3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm 3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3–1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the M r = 91 000 and M r = 22 000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has M r values of 110,000–120,000 and 120,000–135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the M r = 91 000 and M r = 22 000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H 2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carring subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an α-β-type heterodimer composed of a glycosylated M r = 91 000 and non-glycosylated M r = 22 000 polypeptide.