Summary
A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO 2−4 self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl− and HNPG compete for the same site on the anion transporter. The IC50 value for the inhibition of SO 2−4 exchange with HNPG is about 0.13mm at pH 8.0 and 0.36mm at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO 2−4 and chloride exchange but does not modify the apparentK s for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.
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References
Barzilay, M., Ship, S., Cabantchik, Z.I. 1979. Anion transport in red blood cells. I: Chemical properties of anion recognition sites as revealed by structure-activity relationships of aromatic sulfonic acids.Membr. Biochem. 2:277–255
Bennet, V., Steinbuck, P.I. 1980. Association between ankyrin and the cytoplasmic domain of band 3 isolated from the human erythrocyte membrane.J. Biol. Chem. 255:6424–6432
Borders, C.L., Jr., Pearson, L.J., McLaughin, A.E., Gustafson, M.E., Vasiloff, J., An, F.Y., Morgan, D.J. 1979. 4-Hydroxy-3-nitrophenylglyoxal. A chromophoric reagent for arginyl residues in proteinsBiochim.Biophys. Acta 568:491–495
Cabantchik, Z.I., Rothstein, A. 1974. Membrane proteins related to anion permeability of human red blood cells. I: Localization of disulfonic acid binding sites in proteins involved in permeation.J. Membrane Biol. 15:207–226
Falke, J., Chan, S. 1986. Molecular mechanisms of band 3 inhibitors. 1: Transport site inhibitors. 2: Channel blockers. 3: Translocation inhibitors.Biochemistry 25:7888–7906
Fodor, G., Kovács, Ö. 1949. A synthesis of adrenaline-like compounds.J. Am. Chem. Soc. 71:1045–1048
Jennings, M.L., Passow, H. 1979. Anion transport across the erythrocyte membrane, in site proteolysis of band 3 protein and cross-linking of proteolytic fragments by 4,4′-diisothiocyano-dihydrostilbene-2,2′-disulfonate.Biochim. Biophys. Acta 554.
Julien, T., Zaki, L. 1987. New evidence for the essential role of arginine residues in anion transport across the red cell membrane.Biochim. Biophys. Acta 169–174
Knauf, P.A., Ship, S., Breuer, W., McCulloch, L., Rothstein, A. 1978. Asymmetry of the red cell anion exchange system: Different mechanisms of reversible inhibition by N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) at the inside and outside of the membrane.J. Gen. Physiol. 72:607–630
Kopito, R.R., Lodish, H.F. 1985. Primary structure and transmembrane orientations of the murine anion exchange protein.Nature (London) 316:234–238
Passow, H. 1986. Molecular aspects of band 3-protein-mediated anion transport across the red blood cell membrane.Rev. Physiol. Biochem. Pharmacol. 103:63–223
Passow, H., Fasold, H., Gartner, M., Legrum, B., Ruffing, W., Zaki, L. 1980. Anion transport across the red blood cell membrane and the conformation of the protein in band 3.Ann. N.Y. Acad. Sci. 341:361–383
Schnell, K.F. 1972. On the mechanism of inhibition of the sulfate transfer across the human erythrocyte membrane.Biochim. Biophys. Acta 282:265–276
Shaklai, N., Yguerabide, J., Ranney, H.M. 1977. Classification and localization of hemoglobin binding sites on the red blood cell membrane.Biochemistry 16:5593–5597
Shami, Y., Rothstein, A., Knauf, P.A. 1978. Identification of the Cl− transport site of human blood cells by a kinetic analysis of the inhibitory effects of a chemical probe.Biochim. Biophys. Acta 508:357–363
Strapazon, E., Steck, T.L. 1976. Binding at rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.Biochemistry 15:1421–1424
Takahashi, K. 1968. The reaction of phenylglyoxal with arginine residues in proteins.J. Biol. Chem. 243:6171–6179
Wieth, J.O., Bjerrum, P.J., Borders, C.L., Jr. 1982. Irreversible inactivation of red cell chloride exchange with phenylglyoxal, an arginine-specific reagent.J. Gen. Physiol. 79:283–312
Yamasaki, R.B., Vega, A., Feeney, R.E. 1980. Modification of available arginine residues in proteins byp-hydroxyphenyl-glyoxal.Anal. Biochem. 109:32–40
Zaki, L. 1981. Inhibition of anion transport across red blood cells with 1,2-cyclohexandion.Biochem. Biophys. Res. Commun. 99:243–251
Zaki, L. 1982. The effect of arginine specific reagents on anion transport across the red blood cells.Protides Biol. Fluids 29:279–282
Zaki, L. 1983. Anion transport in red blood cells and arginine specific reagents. (1) Effect of chloride and sulfate ions on phenylglyoxal sensitive sites in the red blood cell membrane.Biochem. Biophys. Res. Commun. 110:616–624
Zaki, L. 1984. Anion transport in the red blood cells and arginine specific reagents. The location of14C phenylglyoxal binding sites in the anion transport protein in the membrane of human red cells.FEBS Lett. 169:234–240
Zaki, L., Fasold, H., Schumann, B., Passow, H. 1975. Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells.J. Cell Physiol. 86:471–494
Zaki, L., Julien, T. 1983. Inactivation of the anion transport system in the human red blood cell membrane by a-dicarbonyl reagents.Hoppe-Seyler's Z. Physiol. 364:1233
Zaki, L., Julien, T. 1985a. Anion transport in red blood cells and arginine specific reagents. Interaction between the substrate binding site and the binding site of arginine-specific reagents.Biochim. Biophys. Acta 818:325–332
Zaki, L., Julien, T. 1985b. Inactivation of anion transport across the red blood cells with 4-hydroxy-3-nitrophenylglyoxal.Hoppe-Seyler's Z. Physiol. 366:869 (Abstr.)
Zaki, L., Julien, T. 1986. Chemical properties of anion binding site in red blood cell membrane.8th School on Biophysics of Membrane Transport. School Proceedings, pp. 240–259. Publishing Department of the Agricultural University of Wroclaw, Poland
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Julien, T., Zaki, L. Studies on inactivation of anion transport in human red blood cell membrane by reversibly and irreversibly acting arginine-specific reagents. J. Membrain Biol. 102, 217–224 (1988). https://doi.org/10.1007/BF01925715
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DOI: https://doi.org/10.1007/BF01925715