The topology of the major band 4.5 protein component of the human erythrocyte membrane: characterization of reactive cysteine residues.
A preparation of band 4.5 protein of the red cell membrane, containing largely the sugar transporter, was labelled with the sulfhydryl reagent N-ethyl [14C]maleimide. In preparations denatured with sodium dodecyl sulfate (SDS), all five sulfhydryl groups present in the peptide, Mr 45 000 to 60 000, react with the alkylating agent within 20 min at 37 degrees C. If the peptide is reconstituted in lipid vesicles and cleaved with trypsin before extraction and denaturation with SDS, three sulfhydryl groups are found in a 30 kDa fragment and two in a 19 kDa fragment. In 'native' reconstituted protein only three groups react, even after two hours of exposure, two in the 30 kDa fragment and one in the 19 kDa fragment. Thus, one sulfhydryl group is cryptic, inaccessible to N-ethylmaleimide in each fragment. In intact cells, the single reactive group of the 19 kDa fragment can be protected against reaction with N-ethylmaleimide by the impermeant sulfhydryl reagent, p-chloromercuribenzene sulfonate (PCMBS). It is, therefore, considered to be exposed on the outer face of the membrane. The two reactive groups of the 30 kDa fragment are not protected by PCMBS and are, therefore, not considered to be exposed to the outside medium. Cytochalasin B, a competitive inhibitor of sugar transport affords temporary protection of the exofacial group of the 19 kDa against reaction with N-ethylmaleimide, and affords longer term protection of one of the reactive groups of the 30 kDa fragment. These findings allow conclusions about the topology of the sugar transport protein in the bilayer. Both proteolytic fragments must cross the bilayer. One of three reactive sulfhydryl groups is exofacial and two may be cytoplasmic. The two cryptic groups may be located within the bilayer.
D001798:Blood Proteins; D003545:Cysteine; D003571:Cytochalasin B / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D005033:Ethylmaleimide; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins; D011487:Protein Conformation; D014357:Trypsin
D001798:Blood Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033703:Nucleoside Transport Proteins; D003571:Cytochalasin B; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide