CARBONIC-ANHYDRASE-II BINDS TO THE CARBOXYL-TERMINUS OF HUMAN BAND-3,THE ERYTHROCYTE CL- HCO3- EXCHANGER/

In this study, we provide evidence that the 33-residue carboxyl-termin al (Ct) region of the human erythrocyte chloride/bicarbonate exchanger , band 3, binds carbonic anhydrase II (CAII), Immunofluorescence showe d 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 (K-1/2 = 70 nM) or the me mbrane domain of band 3 (K-1/2 = 100 nM) bound saturably to immobilize d CAII in a solid phase binding assay. The interaction with CAII was s pecifically blocked by an antibody to the Ct of band 3, Affinity blott ing 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 immobilize d CAII was saturable (K-1/2 = 20 nM). The binding rate was slow (t(1/2 ) = 12 h) at physiological ionic strength and pH but was enhanced at l ow ionic strength or acidic pH. Intact band 3 (K-i = 15 nM), the membr ane domain of band 3 (K-i = 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 ch loride/bicarbonate exchanger in red blood cells.