Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte C1-/HCO3- exchanger.

In this study, we provide evidence that the 33-residue carboxyl-terminal (Ct) region of the human erythrocyte chloride/bicarbonate exchanger, band 3, binds carbonic anhydrase II (CAII). Immunofluorescence showed that tomato lectin-mediated clustering of band 3 in ghost membranes caused a similar clustering of CAII, indicating an in situ association. CAII cosolubilized ...
and coimmunoprecipitated with band 3, suggesting that the two proteins form a complex. Band 3 (K1/2 = 70 nM) or the membrane domain of band 3 (K1/2 = 100 nM) bound saturably to immobilized CAII in a solid phase binding assay. The interaction with CAII was specifically blocked by an antibody to the Ct of band 3. Affinity blotting showed that a glutathione S-transferase (GST)-fusion protein (GST-Ct) containing the last 33 residues of human band 3 bound to CAII. The solid phase binding assay showed that binding of GST-Ct to immobilized CAII was saturable (K1/2 = 20 nM). The binding rate was slow (t1/2 = 12 h) at physiological ionic strength and pH but was enhanced at low ionic strength or acidic pH. Intact band 3 (Ki = 15 nM), the membrane domain of band 3 (Ki = 100 nM), or antibodies to the Ct of band 3 were able to block GST-Ct binding to CAII, confirming the specificity of the interaction. Affinity chromatography showed that CAII bound to immobilized GST-Ct with a 1:1 stoichiometry. This work indicates that CAII, the bicarbonate supplier, is directly coupled to band 3, the chloride/bicarbonate exchanger in red blood cells.
Mesh Terms:
Acetazolamide, Anion Exchange Protein 1, Erythrocyte, Bicarbonates, Binding Sites, Carbonic Anhydrase Inhibitors, Carbonic Anhydrases, Chlorides, Chromatography, Affinity, Enzymes, Immobilized, Erythrocyte Membrane, Humans, Peptide Fragments, Protein Binding, Recombinant Fusion Proteins
J. Biol. Chem.
Date: Oct. 23, 1998
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