CHARACTERIZATION OF THE STILBENEDISULPHONATE BINDING-SITE ON BAND-3 MEMPHIS VARIANT-II (PRO-854-]LEU)

Band 3 Memphis variant II is a mutant anion-exchange protein associate d with the Diego a(+) blood group antigen. There are two mutations in this transporter: Lys-56 --> Glu within the cytoplasmic domain, and Pr o-854 --> Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within t he C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS i-isot hiocyanatodihydro-2,2'-stilbenedisulphonate) to 'lysine A' (Lys-539) i n the N-terminal subdomain, suggesting widespread conformational chang es. In this report, we have used various kinetic assays which differen tiate between conformational changes in the two subdomains, to charact erize the stilbenedisulphonate site on band 3 Memphis variant II. We h ave found a significantly higher H2DIDS (a C-terminal-sensitive inhibi tor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'of f' rate constant, but no difference was found between mutant and contr ol when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-termina l-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate or H2DI DS. However, the rate of covalent intrasubunit cross-linking of Lys-53 9 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 --> Leu mutation causes a l ocalized conformational change in the C-terminal subdomain of band 3.