Transport domain of the erythrocyte anion exchange protein.
The anion transport domain of the anion exchange protein (AEP) of human erythrocyte membranes (band 3, 95 kD mol wt) was probed with the substrate and affinity label pyridoxal-5'-phosphate (PLP). Acting from outside, this probe labels two chymotryptic fragments of 65 and 35 kD of AEP but only the 35-kD fragment is protected from labeling by reversibly acting disulfonic stilbenes (DS). It is shown here by functional studies and by immunoblotting with anti-PLP antibodies that transmembrane gradients of anions determine the availability of a 35-kD fragment lys residue to surface labeling by PLP, in analogy with their effects on labeling of 65-kD fragment by DS. On this basis, it is suggested that both fragments contribute to the formation of the transport domain. However, unlike DS, PLP blocks transport when reacted from within released membranes, indicating that the 35-kD fragment might contain components of the mobile unit of the AEP. Using impermeant fluorescence quenchers of PLP of both complexation type (anti-PLP antibodies) or collisional type (acrylamide) as topological probes for PLP-labeled sites, it is deduced that the 65-kD PLP-labeled and the 35-kD PLP-labeled lys groups are inaccessible to macromolecules from either surface, but the 65-kD PLP-lys is accessible to low molecular weight molecules from without while the 35-kD PLP-labeled lys shows accessibility primarily from within the cell surface. The studies indicate that the accommodation of a wide class of anions by AEP might be associated with the flexibility of the transport domain of the protein and its capacity to undergo transport-related conformational changes.
Bar-Noy
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Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Cabantchik
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ZI
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D027321:Anion Transport Proteins; D000838:Anions / Q000097:blood; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D005453:Fluorescence; D006801:Humans; D007120:Immunochemistry; D011732:Pyridoxal Phosphate / Q000276:immunology; D011956:Receptors, Cell Surface / Q000097:blood
D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D000838:Anions; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; C037046:pyridoxal phosphate receptor; D011732:Pyridoxal Phosphate