Erythrocyte band 3 protein: evidence for multiple membrane-crossing segments in the 17 000-dalton chymotryptic fragment.
We have investigated the topology of the band 3 protein of the human erythrocyte membrane by a combination of chemical labeling and proteolytic cleavage. The N-terminal third of the membrane-bound domain of band 3 is a 17 000-dalton chymotryptic fragment that is known to traverse the membrane an odd number of times. At least three lysine residues on this fragment can be labeled by reductive methylation of intact cells, under conditions that cause labeling of exofacial, but not intracellular, lysine residues. One of the labeled lysines is the one that reacts with anionic aryl isothiocyanates, and another is very close to the C terminus of the fragment. Both these are on the C-terminal 11-kilodalton CNBr peptide. The third labeled lysine is on the 6-kilodalton N-terminal CNBr peptide, which had not been previously known to have an extracellular site. Control experiments using a stilbenedisulfonate derivative demonstrate that the labeled 6-kilodalton CNBr peptide is not a degradation product of the 11-kilodalton CNBr fragment. Also, the exofacial lysine on the 6-kilodalton peptide can be labeled by reductive methylation even when the stilbenedisulfonate site is occupied by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate, which blocks band 3 mediated transport of BH4 into the cells. This is further indication that the labeled lysine is accessible from the extracellular water. These data are the first direct evidence that the 17-kilodalton chymotryptic fragment spans the membrane more than once.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D004910:Erythrocyte Membrane / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments
D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid