Synthesis and Characterization of a Novel Spin-Labeled Affinity Probe of Human Erythrocyte Band 3:  Characteristics of the Stilbenedisulfonate Binding Site†

Biochemistry ◽  
1996 ◽  
Vol 35 (21) ◽  
pp. 6931-6943 ◽  
Author(s):  
Douglas J. Scothorn ◽  
Walter E. Wojcicki ◽  
Eric J. Hustedt ◽  
Albert H. Beth ◽  
Charles E. Cobb
Keyword(s):  
Binding Site ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Synthesis And Characterization ◽  
Affinity Probe

scholarly journals Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 932-938 ◽  
Author(s):  
Haiyan Chu ◽  
Andrew Breite ◽  
Peter Ciraolo ◽  
Robert S. Franco ◽  
Philip S. Low
Keyword(s):  
Binding Site ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Major Protein ◽  
Human Erythrocyte Membrane ◽  
Functional Studies ◽  
High Sequence Homology ◽  
Affinity Interaction ◽  
Related Proteins

Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates cellular properties performed by these and/or related proteins. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 reversibly with high affinity, these observations raise the hypothesis that hemoglobin might regulate erythrocyte properties through its reversible, oxygenation-dependent association with band 3. To explore this hypothesis, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that (1) deoxyHb binds to residues 12-23 of band 3; (2) mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction would have been possible had it been beneficial for survival; (3) Hb does not bind to 2 other sequences in band 3 despite their high sequence homology to residues 12-23, and (4) the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by the oxygenation state of the cell.

scholarly journals Identification and characterization of a second 4,4′-dibenzamido-2,2′-stilbenedisulphonate (DBDS)-binding site on band 3 and its relationship with the anion/proton co-transport function

2005 ◽  
Vol 388 (1) ◽  
pp. 343-353 ◽  
Author(s):  
James M. SALHANY ◽  
Karen S. CORDES ◽  
Renee L. SLOAN
Keyword(s):  
Binding Site ◽  
Proton Transport ◽  
Chloride Concentration ◽  
Band 3 ◽  
Low Ph ◽  
Binding Mechanism ◽  
Access Channel ◽  
Binding Kinetic ◽  
Transport Site

Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (∼5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4′-dibenzamido-2,2′-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0↔T0+D↔TD↔RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 μM and a T0+D↔TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5±11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27±1.27 μM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.

scholarly journals The aldolase-binding site of the human erythrocyte membrane is at the NH2 terminus of band 3.

1981 ◽  
Vol 256 (21) ◽  
pp. 11203-11208 ◽  
Author(s):  
S.N. Murthy ◽  
T. Liu ◽  
R.K. Kaul ◽  
H. Köhler ◽  
T.L. Steck
Keyword(s):  
Binding Site ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Band 3 ◽  
Human Erythrocyte Membrane

scholarly journals Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)

1996 ◽  
Vol 317 (2) ◽  
pp. 509-514 ◽  
Author(s):  
James M. SALHANY ◽  
Renee L. SLOAN ◽  
Lawrence M. SCHOPFER
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Covalent Binding ◽  
Band 3 ◽  
Blood Group Antigen ◽  
Cross Linking ◽  
Anion Exchange Protein ◽  
Membrane Bound ◽  
And Control

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

scholarly journals Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 inEscherichia coli

1992 ◽  
Vol 1 (9) ◽  
pp. 1206-1214 ◽  
Author(s):  
Cheng C. Wang ◽  
Ryuichi Moriyama ◽  
Philip S. Low ◽  
Janine A. Badylak ◽  
Jack E. Dixon ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Inescherichia Coli ◽  
Purification And Characterization

Freeze-fracture Electron Microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

1990 ◽  
Vol 48 (3) ◽  
pp. 524-525 ◽  
Author(s):  
Gheorghe Benga ◽  
Anthony Brain ◽  
Victor I. Pop ◽  
John Wrigglesworth
Keyword(s):  
Human Erythrocyte ◽  
Freeze Fracture ◽  
Band 3 ◽  
Erythrocyte Membranes ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
Water Permeation ◽  
Human Erythrocyte Membranes ◽  
Erythrocyte Membrane Proteins

The intra-membrane particles (IMPs) observed on the fracture face of frozen erythrocyte membranes are thought to correspond primarily to “band 3” tetramers or dimers. Some recent studies correlating the inhibition of water diffusion in erythrocytes by p-chloromercuribenzene sulfonate (PCMBS) with the binding of 203Hg to erythrocyte membrane proteins has enabled band 3 and the polypeptides in band 4.5 to be identified as the proteins associated with the channels for water permeation in human erythrocytes. A further characterization of the effects of the incubation of human erythrocyte membranes with PCMBS and N-ethylmaleimide (NEM) has been performed as previously described. Experimental conditions have been previously described.A comparison was made of the appearance of freeze-etched membranes of control erythrocytes and erythrocytes with the sulphydryl reagents. It was found that on many of the control and NEM-treated cells, small (50-100 nm) elevated patches could be seen (Fig. 1, 2 and 3). These are present on both fracture and etch faces and are devoid of any intramembrane particles. The patch elevations were never observed in the membranes of PCMBS-treated cells (Fig. 4).

ChemInform Abstract: Synthesis and Characterization of Hydrogen-Bonded Assemblies: Toward the Generation of Binding Site Diversity

ChemInform ◽  
2010 ◽  
Vol 30 (49) ◽  
pp. no-no
Author(s):  
D. N. Reinhoudt ◽  
P. Timmerman ◽  
F. Cardullo ◽  
M. Crego-Calama
Keyword(s):  
Binding Site ◽  
Synthesis And Characterization ◽  
Site Diversity ◽  
Hydrogen Bonded
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