Stoichiometry of a half-turnover of band 3, the chloride transport protein of human erythrocytes.

The kinetics of human red blood cell Cl transport have been studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4'-dinitro-2,2'-stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per ghost, which is abolished by pretreatment with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), appears to represent the Cl that is transported during the first half-turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.

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THE STUDY OF THE ANION TRANSPORT PROTEIN (‘BAND 3 PROTEIN’) IN THE RED CELL MEMBRANE BY MEANS OF TRITIATED 4,4′-DIISOTHIOCYANO-DIHYDROSTILBENE-2,2-DISULFONIC ACID (3H2DIDS)

Chloride Transport in Biological Membranes ◽

10.1016/b978-0-12-775280-8.50005-6 ◽

1982 ◽

pp. 1-31 ◽

Cited By ~ 4

Author(s):

H. Passow ◽

H. Fasold ◽

M.L. Jennings ◽

S. Lepke

Keyword(s):

Cell Membrane ◽

Protein Band ◽

Band 3 ◽

Anion Transport ◽

Transport Protein ◽

Disulfonic Acid ◽

Red Cell ◽

Band 3 Protein ◽

Red Cell Membrane

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Kinetics of chloride transport across fish red blood cell membranes

Journal of Experimental Biology ◽

10.1242/jeb.198.10.2237 ◽

1995 ◽

Vol 198 (10) ◽

pp. 2237-2244 ◽

Cited By ~ 4

Author(s):

F Jensen ◽

J Brahm

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Atlantic Cod ◽

Chloride Transport ◽

Band 3 ◽

Transport Kinetics ◽

Human Values ◽

Cl Transport ◽

Kinetics Of

The continuous flow tube method was used to investigate the kinetics of chloride transport, and its potential oxygenation-dependency, in red blood cells (RBCs) from four teleost fish species and man. A significant interspecific variation in Cl- transport kinetics was found. At 15 °C, the rate constant k for unidirectional 36Cl- efflux was significantly lower in RBCs from eel and carp than in RBCs from rainbow trout and Atlantic cod. The values of k of cod RBCs at 15 °C and of human RBCs at 37 °C were not significantly different. The volume and surface area of the RBCs were evaluated and used to calculate the apparent membrane permeability to Cl- (PCl). The magnitude of PCl followed the sequence: eel<carp<trout¾cod. PCl values in trout and cod at 15 °C were similar to human values at 37 °C. An extrapolation of human values to 15 °C revealed that the Cl- shift at this temperature was considerable faster in all four teleosts than in man. This illustrates appropriate adaption of band-3-mediated anion transport to the different temperature regimes encountered by fish and mammals. The Cl- transport kinetics did not differ significantly between oxygenated and deoxygenated RBCs in any of the species examined. The apparent absence of any effect of a change in haemoglobin oxygen-saturation may be related to the presence of a flexible link which results in minimal interaction between the membrane domain (mediating Cl- transport) and the cytoplasmic domain (to which oxygenation-dependent haemoglobin binding occurs) of band 3. In carp, Cl- transport kinetics were not influenced by pH over the extracellular pH (pHe) range 7.68.36, which spans the in vivo pHe range. The data are discussed in relation to the rate-limiting role of red blood cell HCO3-/Cl- exchange for CO2 excretion.

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Eosin-5-maleimide inhibits red cell Cl- exchange at a noncompetitive site that senses band 3 conformation

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.5.c1144 ◽

1993 ◽

Vol 264 (5) ◽

pp. C1144-C1154 ◽

Cited By ~ 9

Author(s):

P. A. Knauf ◽

N. M. Strong ◽

J. Penikas ◽

R. B. Wheeler ◽

S. Q. Liu

Keyword(s):

Inhibitory Concentration ◽

Potent Inhibitor ◽

Chloride Concentration ◽

Competitive Inhibitor ◽

Band 3 ◽

Reversible Inhibitor ◽

Red Cell ◽

Inhibitory Potency ◽

A Site ◽

Transport Site

Eosin-5-maleimide (EM) has been used as a fluorescent probe for the external-facing transport site of the human erythrocyte band 3 protein. Changes in chloride concentration at both sides of the membrane have no significant effect on the inhibitory potency of EM as a reversible inhibitor of Cl- exchange at 0 degrees C, however, demonstrating that it is not a competitive inhibitor. The affinity of EM for the form of band 3 with the transport site facing outward is approximately five times greater than for the form with the transport site facing the cytoplasm; binding of iodide to the external transport site causes no statistically significant decrease in affinity for EM. Eosin, without the maleimide moiety, is a slightly more potent inhibitor than is EM. Erythrosin, an analogue with four iodide atoms replacing the four bromide atoms in eosin, is a much more potent inhibitor, with a half-inhibitory concentration of only 3.1 microM, > 30 times lower than that of EM. Neither eosin nor erythrosin inhibition is affected by changes in chloride concentration as would be expected for a competitive inhibitor. Thus EM and the other eosin derivatives bind to a site separate from the external transport site, but one that is affected by the changes of transport site conformation from the inward-facing to the outward-facing state.

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Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

The Journal of General Physiology ◽

10.1085/jgp.81.4.453 ◽

1983 ◽

Vol 81 (4) ◽

pp. 453-484 ◽

Cited By ~ 60

Author(s):

P J Bjerrum ◽

J O Wieth ◽

C L Borders

Keyword(s):

Intracellular Ph ◽

Covalent Binding ◽

Chloride Concentration ◽

Protein Band ◽

Band 3 ◽

Anion Transport ◽

Transport Protein ◽

Red Cell ◽

Red Cell Membrane ◽

Arginyl Residues

The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal: arg stoichiometry of 2:1. Indiscriminate modification of all arg in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were above 10. Selective modification of extracellularly exposed arg was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of four phenylglyoxals per band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of two phenylglyoxals per band 3, which suggests that transport function is inactivated by the modification of a single arg. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. In contrast, the primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the second cleavage product, a 65,000-dalton fragment. This finding supports the view that the transport region of band 3 is composed of strands from both chymotryptic fragments. The binding of phenylglyoxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by a reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 10 micrometers, only 50% of the band 3 molecules were labeled with [3H]-DIDS during 90 min at 38 degrees C, thereby demonstrating an interaction between binding of the two inhibitors to the protomers of the oligomeric band 3 molecules.

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