Transport-related conformational states of the band 3 protein: probing with 1-fluoro-2,4-dinitrobenzene.

The present article provides experimental evidence for previous claims, that Lys 539, without being directly involved in anion binding or translocation, is allosterically linked to the anion binding sites of the band 3 protein and to some other, as yet unidentified amino acid residue. The evidence is based on a detailed study of the kinetics of inhibition of sulphate equilibrium exchange by 1-fluoro-2,4-dinitrobenzene (N2ph-F). It is shown that the mutation of Lys 558 in mouse band 3, which is homologous to Lys 539 in human band 3, renders the transport protein insusceptible to inhibition by N2pH-F, confirming that it is the modification of this residue which results in the inhibition of band 3-mediated transport. The investigation of the kinetics of the modification of human band 3 revealed that the modification is not preceded by non-covalent N2ph-F binding and hence governed by the structure of the native protein near Lys 539. In chloride-containing media, the rate constant of dinitrophenylation of Lys 539 is about 15 times higher than in sulphate-containing media. This suggests that the chemical nature of the anion species bound to band 3 determines whether Lys 539 exists in a buried or exposed state and hence represents a reporter group which characterizes the functional state of the transport protein. The parameter values describing the effects of anion binding on the interactions between Lys 539 and an allosterically linked, unidentified amino acid residue were determined by means of a mathematical model which permitted the quantitative evaluation of the data.

Anion transport across the red blood cell membrane and the conformation of the protein in Band 3.

Measuring the rate of dinitrophenylation of a specific lysine residue (called a) that is allosterically linked to the transfer site, it could be demonstrated that the anion transport protein may exist in two different conformational states, designated cis and trans. In the cis conformation a is easily accessible for reaction with dinitrofluorobenzene; in the trans conformation, a is less accessible. In the presence of the substrate anion Cl, the equilibrium between the cis and trans conformation is towards the cis conformation. Reversibly acting inhibitors of anion transport arrest the transport system, either predominantly in the cis or in the trans conformation. Phlorizin and certain positively charged derivatives of furosemide produce arrest in cis conformation, internal 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonate (APMB) and Ca++ in trans conformation. Within this frame of reference, the different susceptibilities of the transfer site to internal and external 4,4' diacetamido-2,2'-stilbene disulfonate (DAS) are interpreted on the assumption that the conformation of the transfer site changes during the transition of the transport protein from the cis to the trans conformation, so that in the trans conformation a reaction with DAS is no longer possible.