Nucleoside transporter of pig erythrocytes. Kinetic properties, isolation and reaction with nitrobenzylthioinosine and dipyridamole.
Rapid kinetic techniques were used to measure the transport of uridine in pig erythrocytes in zero-trans entry and exit and equilibrium exchange protocols. The kinetic parameters were computed by fitting appropriate integrated rate equations to the time-courses of transmembrane equilibration of radiolabeled uridine. Transport of uridine conformed to the simple carrier model with directional symmetry, but differential mobility of substrate-loaded and empty carrier. At 5 degrees C, the carrier moved about 30-times faster when loaded than when empty. Uridine transport was inhibited in a concentration-dependent manner by nitrobenzylthioinosine and dipyridamole and the inhibition correlated with the binding of the inhibitors to high-affinity binding sites on the cells (Kd about 1 and 10 nM, respectively). Thus, in its kinetic properties, differential mobility when empty and loaded, and sensitivity to inhibition by nitrobenzylthioinosine and dipyridamole, the transporter of pig erythrocytes is very similar to that of human erythrocytes. Also, the total number of high-affinity binding sites for nitrobenzylthioinosine and dipyridamole/cell were similar for the two cell types and the [3H]nitrobenzylthioinosine-labeled carrier of pig erythrocytes, just as that of human red cells, was mainly recovered in the band 4.5 protein fraction of Triton X-100-solubilized membranes. However, sodium dodecylsulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled band 4.5 membrane proteins indicated a slightly higher molecular weight for the transporter from pig than human erythrocytes. We have also confirmed the lack of functional sugar transport in erythrocytes from adult pigs by measuring the uptake of various radiolabeled sugars. But in spite of the lack of functional sugar transport we recovered as much band 4.5 protein from pig as from human erythrocyte membranes.
D000345:Affinity Labels; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002241:Carbohydrates / Q000097:blood; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D004176:Dipyridamole / Q000097:blood* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007288:Inosine / Q000031:analogs & derivatives*; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D033703:Nucleoside Transport Proteins; D010777:Photochemistry; D013552:Swine; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000097:blood / Q000494:pharmacology; D014529:Uridine / Q000097:blood
D000345:Affinity Labels; D002241:Carbohydrates; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; D007288:Inosine; D004176:Dipyridamole; C001789:4-nitrobenzylthioinosine; D014529:Uridine