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Abstract:
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.
