Pepsin cleavage of band 3 produces its membrane-crossing domains.
After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) or with N-ethylmaleimide, probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N-ethylmaleimide. Because the location of the N-ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D004910:Erythrocyte Membrane / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010434:Pepsin A; D005033:Ethylmaleimide; D003488:Cyanogen Bromide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid