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.
Vince
J W
JW
Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto M5S 1A8, Canada.
Reithmeier
R A
RA
D000086:Acetazolamide / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized; D004910:Erythrocyte Membrane / Q000737:chemistry; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D002712:Chlorides; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D002256:Carbonic Anhydrases; D000086:Acetazolamide