© 2006 The Japanese Biochemical Society.
Regular Paper |
The Functional Role of Arginine 901 at the C-Terminus of the Human Anion Transporter Band 3 Protein
1 Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, and 2 Department of Immunology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582
* To whom correspondence should be addressed at: Department of Immunology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582. Tel: +81-92-642-6663, Fax: +81-92-642-6667, E-mail: abe{at}phar.kyushu-u.ac.jp
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.