Interaction among anion, cation and glucose transport proteins in the human red cell.
The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.
Janoshazi
A
A
Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 02115.
Solomon
A K
AK
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000097:blood; D027321:Anion Transport Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000097:blood*; D002712:Chlorides / Q000097:blood; D003571:Cytochalasin B / Q000097:blood; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D010042:Ouabain / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000097:blood
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D009004:Monosaccharide Transport Proteins; D013431:Sulfates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D003571:Cytochalasin B; D010042:Ouabain; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid