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キーワード:
band 3; expression; anion transport; red cell; Xenopus oocytes; mutagenesis; stilbene disulfonates
要旨:
The effect of mutation of either Lys 558 or Lys 869 or both on mouse erythroid band 3 protein (AE1)-mediated 36Cl- efflux and its inhibition by pyridoxal 5-phosphate (P5-P), DNDS and H2DIDS were studied. Regardless of the mutation, band 3 was always capable of executing Cl- self-exchange. P5-P (5 mM, pH 7.6) produced irreversible inhibition in the wild type (KK) and in the mutant in which Lys 558 (NK) or Lys 869 (KM) had been replaced by asparagine (N) or methionine (M), respectively. However, when both residues were replaced, mutant (NM), irreversible inhibition could no longer be achieved. This shows that P5-P is capable of producing inhibition with either one of the lysine residues, 558 or 869. Inhibition by DNDS changed dramatically upon mutation. The Ki app increased from 6.0 microM in the wild type (KK) to 23 microM in the mutant NK, to 73 microM in the mutant KM and to 474 microM in the double mutant NM. The Km value for activation of the transport system by varying the substrate concentration by isosmotic substitution of Cl- with SO42- decreased from 42 mM in the wild type (KK) to 11.3 mM in the mutant NM. The results show that both Lys 558 and Lys 869 are involved in the maintenance of the structure of the overlapping binding sites for stilbene disulfonates and the substrate Cl-. In the double mutant NM, H2DIDS is no longer able to produce irreversible inhibition at pH 7.6. This is evidently related to the replacement of Lys 558 (pK 8.2) by Asn 558 in this mutant (see Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., Passow, H., 1989. EMBO J. 8:3601-3609). However, at pH 9.5, some irreversible inhibition could still be observed. This suggests that the other lysine residue (pK 10.8) that is known to be involved in covalent binding with the second isothiocyanate group of H2DIDS is still present, and hence, not identical to Lys 869, which had been substituted by a methionine residue. However, this result remains inconclusive since after mutagenesis, the H2DIDS may produce inhibition at a site that is not normally involved in H2DIDS binding.
