title: Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3.
abstract: Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8.
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kojima
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Kawaguchi
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Shimada
authors.affiliation: Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Aso
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D006801:Humans; D008239:Lysine / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010206:Papain / Q000494:pharmacology*; D010446:Peptide Fragments / Q000187:drug effects*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D010446:Peptide Fragments; D010206:Papain; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
keywords: DIDS; band 3; erythrocyte; fluorescence; papain