Interaction between red cell membrane band 3 and cytosolic carbonic anhydrase.
We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.
Kifor
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Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 02115.
Toon
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MR
Janoshazi
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Solomon
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis / Q000378:metabolism*; D001557:Benzenesulfonates / Q000378:metabolism / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000032:analysis / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000201:enzymology*; D003619:Dansyl Compounds / Q000378:metabolism / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000201:enzymology; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D013449:Sulfonamides / Q000378:metabolism / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D001557:Benzenesulfonates; D003619:Dansyl Compounds; D013449:Sulfonamides; D002256:Carbonic Anhydrases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid