Evidence from electron paramagnetic resonance for function-related conformation changes in the anion-transport protein of human erythrocytes.
The erythrocyte membrane protein involved in anion transport (band 3) was isolated in its native lipid milieu in the form of leaky vesicles and then was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-meleimide (MalMe4PipO). The resulting electron paramagnetic resonance spectrum of band-3-bound MalMe4PipO was resolved into a rapid tumbling component and another, relatively immobile component. The percentage of the signal contributed by the mobile component (Q), was sensitive to various characteristic factors known to affect erythrocyte anion transport: Q was a hyperbolic function of chloride concentration displaying a half-saturation constant K1/2 similar to that of chloride transport. On the other hand Q showed a biphasic response to sulfate concentration, in line with the relatively high affinity of sulfate for the anion modifier site. Q was a saturable function of pH, either in presence of Cl- or SO4(-2), showing a pKa between pH 6.0 and 6.5, in analogy with the pH titration curve of Cl- and SO4(-2), transport. Spin-labelled vesicles treated with a covalent inhibitor of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, were markedly less susceptible to changes in Cl- concentration. It is suggested that the electron paramagnetic resonance spectrum of MalMe4PipO covalently bound to the band-3 protein, reports conformational changes which are related to the anion-transport function of this protein.
D027321:Anion Transport Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000097:blood; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005033:Ethylmaleimide; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013431:Sulfates / Q000097:blood
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D008565:Membrane Proteins; D013431:Sulfates; D005033:Ethylmaleimide