A structural study of the membrane domain of band 3 by tryptic digestion: Conformational change of band 3 in situ induced by alkali treatment

Dongchon Kang, Kenshi Okubo, Naotaka Hamasaki, Naotaka Kuroda, Hiroshi Shiraki

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53 Citations (Scopus)


Nine peptides derived from the transmembrane domain of band 3 were purified and sequenced. All of the sequences agreed completely with deduced sequences from cDNA of human erythroid band 3. Five peptides, KS-1 to KS-5, were released from the band 3 molecule when alkali-stripped membranes were digested with trypsin, while four other peptides, KM-6 to KM-9, were obtained following subsequent urea treatment. This indicates that at least 13 new in situ cleavage sites were demonstrable by these procedures, that the released peptides are parts of hydrophilic connector loops, and that the other peptide portions constitute membrane-spanning helices. The topological designations are consistent with the hydropathy prediction of murine band 3 according to Passow ((1986) Rev. Physiol. Biochem. Pharmacol. 103, 61-203). One mol of histidine residue was found/mole of KS-1, KS-2, KS-4, and KM-6. The conformation of band 3 in situ was apparently changed by alkali treatment of erythrocyte membranes, i.e. the amount of KS-1, KS-2, and KS-4 peptides released by trypsin treatment increased as NaOH concentration was raised from 10 to 100 mM. Similarly, [3H]dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonic acid was found to bind to band 3 in membranes treated with 10 mM NaOH as well as to band 3 in white ghosts, but not to membranes treated with 100 mM NaOH. In addition, alkali treatment of membranes tended to increase the amount of band 3 cross-linked by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The conformational change in band 3 by alkali treatment was also supported by the interaction of antibodies against peptides released by trypsin. The release of KS-1, KS-2, and KS-4 from the membrane was strongly inhibited by pretreating the erythrocyte membrane with DIDS, suggesting that the DIDS-band 3 complex which is in the outward facing form, is more compact and becomes resistant to trypsin compared to band 3 without DIDS.

Original languageEnglish
Pages (from-to)19211-19217
Number of pages7
JournalJournal of Biological Chemistry
Issue number27
Publication statusPublished - Sept 25 1992
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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