TY - JOUR
T1 - The interaction of an anionic photoreactive probe with the anion transport system of the human red blood cell
AU - Cabantchik, Z. Ioav
AU - Knauf, Philip A.
AU - Ostwald, Thomas
AU - Markus, Howard
AU - Davidson, Lorinda
AU - Breuer, William
AU - Rothstein, Aser
PY - 1976/12/2
Y1 - 1976/12/2
N2 - N-4-azido-2-nitrophenyl)-2-aminoethyl[35S]sulfonate is employed as a photoreactive probe for the anion transport system in the human erythrocyte. In the dark and at 37 °C the probe penetrates the membrane via a pathway sensitive to specific inhibitors of anion permeability. It reversibly inhibits sulfate and chloride fluxes but the inhibition is reduced by higher concentrations of sulfate. Upon photolysis to produce a reactive nitrene (at 0 °C to minimize penetration), the probe reacts covalently with outer membrane components resulting in an irreversible inhibition of anion permeability. Under appropriate conditions the degree of inhibition after photoactivation (irreversible) is almost the same as that in the dark (reversible). The binding sites for the radioactive probe are largely found in proteins of 95 000 apparent molecular weight (band 3). After pronase treatment of the labelled cells, most of the probe is found in a 65 000 molecular weight segment derived from the 95 000 molecular weight protein. In this respect the photoreactive probe resembles anoher potent irreversible inhibitor of anion transport, 4, 4′-diisothiocyano-2, 2′ stilbene disulfonate. In fact, most of the binding sites for each probe are common to both. Thus, in the dark, the azido derivative protects the anion system from inhibition by DIDS and substantially reduces the binding of DIDS to band 3 protein. Conversely, pretreatment with DIDS substantially reduces the binding of the photoreactive probe to the same protein. The fact that an apparent substrate for the anion permeation system competes for binding sites with a specific non-penetrating inhibitor of anion permeability suggests that the inhibitory and transport sites may be closely related and implicates the 95 000 molecular weight protein as the element of the anion transport system which contains the substrate binding site.
AB - N-4-azido-2-nitrophenyl)-2-aminoethyl[35S]sulfonate is employed as a photoreactive probe for the anion transport system in the human erythrocyte. In the dark and at 37 °C the probe penetrates the membrane via a pathway sensitive to specific inhibitors of anion permeability. It reversibly inhibits sulfate and chloride fluxes but the inhibition is reduced by higher concentrations of sulfate. Upon photolysis to produce a reactive nitrene (at 0 °C to minimize penetration), the probe reacts covalently with outer membrane components resulting in an irreversible inhibition of anion permeability. Under appropriate conditions the degree of inhibition after photoactivation (irreversible) is almost the same as that in the dark (reversible). The binding sites for the radioactive probe are largely found in proteins of 95 000 apparent molecular weight (band 3). After pronase treatment of the labelled cells, most of the probe is found in a 65 000 molecular weight segment derived from the 95 000 molecular weight protein. In this respect the photoreactive probe resembles anoher potent irreversible inhibitor of anion transport, 4, 4′-diisothiocyano-2, 2′ stilbene disulfonate. In fact, most of the binding sites for each probe are common to both. Thus, in the dark, the azido derivative protects the anion system from inhibition by DIDS and substantially reduces the binding of DIDS to band 3 protein. Conversely, pretreatment with DIDS substantially reduces the binding of the photoreactive probe to the same protein. The fact that an apparent substrate for the anion permeation system competes for binding sites with a specific non-penetrating inhibitor of anion permeability suggests that the inhibitory and transport sites may be closely related and implicates the 95 000 molecular weight protein as the element of the anion transport system which contains the substrate binding site.
UR - http://www.scopus.com/inward/record.url?scp=0017100532&partnerID=8YFLogxK
U2 - 10.1016/0005-2736(76)90322-9
DO - 10.1016/0005-2736(76)90322-9
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C2 - 999926
AN - SCOPUS:0017100532
SN - 0005-2736
VL - 455
SP - 526
EP - 537
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 2
ER -