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.

Localization and Functional Characterization of the Deoxyhemoglobin Binding Site on Human Erythrocyte Band 3.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 140-140 ◽  
Author(s):  
Haiyan Chu ◽  
Andrew Breite ◽  
Peter Ciraolo ◽  
Robert S. Franco ◽  
Philip S. Low
Keyword(s):  
Ion Transport ◽  
Binding Site ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Functional Characterization ◽  
Band 3 ◽  
Major Protein ◽  
Functional Studies ◽  
Reversible Association ◽  
Affinity Interaction

Abstract Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins of the cell. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates metabolic, ion transport and mechanical properties of the cell. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 with high affinity, these observations raise the hypothesis that hemoglobin (Hb) might regulate erythrocyte properties through its oxygenation-dependent association with band 3. To explore how reversible association of deoxyHb with band 3 might modulate erythrocyte properties, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that: the deoxyHb binding site on the erythrocyte membrane comprises residues 12–23 of band 3; 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 was possible had it been beneficial; Hb does not bind to two similar sequences in band 3 despite their high sequence homology to residues 12–23, and 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 Hb oxygenation. We conclude that nature has evolved a deoxyHb binding site on band 3 whose affinity is regulated by O2, enabling the reversible association of deoxyHb with band 3 to serve as a regulator of the oxygenation-dependent properties of the cell.

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

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 Mapping of glycolytic enzyme-binding sites on human erythrocyte band 3

2006 ◽  
Vol 400 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Haiyan Chu ◽  
Philip S. Low
Keyword(s):  
Binding Site ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Band 3 ◽  
Glycolytic Enzyme ◽  
Enzyme Complex ◽  
Molecular Architecture ◽  
Human Erythrocyte Membrane ◽  
Enzyme Binding

Previous work has shown that GAPDH (glyceraldehyde-3-phosphate dehydrogenase), aldolase, PFK (phosphofructokinase), PK (pyruvate kinase) and LDH (lactate dehydrogenase) assemble into a GE (glycolytic enzyme) complex on the inner surface of the human erythrocyte membrane. In an effort to define the molecular architecture of this complex, we have undertaken to localize the binding sites of these enzymes more accurately. We report that: (i) a major aldolase-binding site on the erythrocyte membrane is located within N-terminal residues 1–23 of band 3 and that both consensus sequences D6DYED10 and E19EYED23 are necessary to form a single enzyme-binding site; (ii) GAPDH has two tandem binding sites on band 3, located in residues 1–11 and residues 12–23 respectively; (iii) a PFK-binding site resides between residues 12 and 23 of band 3; (iv) no GEs bind to the third consensus sequence (residues D902EYDE906) at the C-terminus of band 3; and (v) the LDH- and PK-binding sites on the erythrocyte membrane do not reside on band 3. Taken together, these results argue that band 3 provides a nucleation site for the GE complex on the human erythrocyte membrane and that other components near band 3 must also participate in organizing the enzyme complex.

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.

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