PubMed · 5904371
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D L Alvis. 1966. Electroretinographic changes in controlled chronic open-angle glaucoma.. https://doi.org/10.1016/0002-9394(66)90756-2
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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.
Carbonic anhydrase V (CA-V) is a mitochondrial enzyme that provides bicarbonate for pyruvate carboxylase in liver and kidney. In the course of a survey of the tissue distribution of CA-V, we detected intense immunostaining in pancreatic islets when sections from rat and mouse pancreases were reacted with a polyclonal antibody to recombinant mouse CA-V. The distribution and large number of CA-V-positive cells in each islet suggested that they represented beta cells. Double immunofluorescence staining of tissue sections and isolated islet cells showed cellular colocalization of CA-V and insulin, confirming that beta cells contain CA-V. Western blotting of rat islets of Langerhans and primary beta cells showed 33- and 30-kDa polypeptides of precursor and mature CA-V, respectively. The CA-V expression was beta cell-specific since no CA-V immunoreaction was detected in the primary alpha cells. Immunohistochemical staining for CA-I, CA-II, CA-IV, CA-VI, and CA-IX was negative in beta cells, and Western blotting of beta cells also failed to identify any CA in beta cells except CA-V. The specific localization of CA-V in beta cells led us to hypothesize that CA-V may be functionally linked to the regulation of insulin secretion. Consistent with this hypothesis, the CA inhibitor acetazolamide was found to be a strong inhibitor of glucose-stimulated insulin secretion by isolated rat pancreatic islets.
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