TY - JOUR
T1 - The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein
AU - Takazaki, Shinya
AU - Abe, Yoshito
AU - Kang, Donchon
AU - Li, Chunyan
AU - Jin, Xiuri
AU - Ueda, Tadashi
AU - Hamasaki, Naotaka
N1 - Funding Information:
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to Y. A. and N. H., and the Kaibara Foundation to Y. A. We are grateful to Professor Michael J. A. Tanner (Department of Biochemistry, School of Medical Sciences,
PY - 2006/5
Y1 - 2006/5
N2 - To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.
AB - To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.
UR - http://www.scopus.com/inward/record.url?scp=33745738625&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745738625&partnerID=8YFLogxK
U2 - 10.1093/jb/mvj097
DO - 10.1093/jb/mvj097
M3 - Article
C2 - 16751598
AN - SCOPUS:33745738625
VL - 139
SP - 903
EP - 912
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 5
ER -