pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue
| Autor: | Daniel Kietz, Detlef Bartel, Hermann Passow, Sigrid Lepke |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
4-Acetamido-4'-isothiocyanatostilbene-2
2'-disulfonic Acid Stereochemistry Xenopus Biophysics 4 4'-Diisothiocyanostilbene-2 2'-Disulfonic Acid Biochemistry chemistry.chemical_compound Mice Chlorides Anion Exchange Protein 1 Erythrocyte Animals Bovine serum albumin Binding site Lipid bilayer Band 3 biology Lysine Erythrocyte Membrane Temperature Cell Biology Membrane transport Hydrogen-Ion Concentration Inorganic anion transport Kinetics chemistry DIDS Covalent bond biology.protein Mutagenesis Site-Directed Oocytes Asparagine Oligonucleotide Probes |
| Zdroj: | Biochimica et biophysica acta. 1064(1) |
| ISSN: | 0006-3002 |
| Popis: | The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H 2 DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b . In the red cell membrane, the p K value of Lys a is about 2.5 p K units lower than the p K value of Lys b . Hence the susceptibility of Lys a to irreversible modification by H 2 DIDS far exceeds the susceptibility to Lys b . In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl − exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H 2 DIDS at a concentration (20 μM) which saturates all H 2 DIDS binding sites with reversibly bound H 2 DIDS ( K I = 0.3 μ M and 1.1. μ M, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H 2 DIDS was replaced by bovine serum albumin to remove free and reversibly bound H 2 DIDS from the extracellular phase. Subsequent measurements of Cl − efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H 2 DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, p K values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H 2 DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single p K value of 8.3. In the mutant, Lys a is missing. Hence, covalent reaction can only take place with Lys b . Measurements over the appropriate pH range could be described by a single p K of 10.8. These values are 0.8–0.9 p K units higher than those previously obtained in experiments with band 3 in the red cell membrane (Kampmann et al. (1982) J. Membr. Biol. 70, 199–216). The difference can be accounted for by the difference of temperature which amounted to 20°C in the present experiments with the oocytes and to 30°C in the previous work with red blood cells. The results suggest that location and function of Lys a and Lys b are essentially the same after expression in the lipid bilayer of the red blood cell and the oocyte. |
| Databáze: | OpenAIRE |
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