Abstract
Nitrobenzylthioinosine (NBMPR) was employed as a covalent probe of the erythrocyte nucleoside transporter. This nucleoside analogue, a potent inhibitor of nucleoside transport, binds tightly (KD = 10(-10) - 10(-9) M) but reversibly to specific sites on the carrier mechanism. High intensity UV irradiation of intact human erythrocytes, isolated "ghosts," and "protein-depleted" membranes in the presence of [3H]NBMPR and dithiothreitol (as a free radical scavenger) under nonequilibrium and equilibrium binding conditions resulted in selective covalent incorporation of 3H into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr = 45,000-65,000). Covalent labeling of band 4.5 protein(s) under equilibrium binding conditions was inhibited by nitrobenzylthioguanosine, dipyridamole, uridine, and adenosine. A similar photolabeling pattern was observed using membranes from pig erythrocytes. In contrast, no incorporation of radioactivity into band 4.5 was observed under equilibrium binding conditions with membranes from nucleoside-impermeable sheep erythrocytes. These experiments suggest that the human and pig erythrocyte nucleoside transporters are band 4.5 polypeptides, a conclusion supported by previous isolation studies based on the assay of reversible [3H]NBMPR binding activity.
Highlights
M) but reversibly to specific siteson the carrier mechanism
The same impermeable sheep erythrocytes. These experiments series of experiments established that NBMPR itself could suggest that the human and pig erythrocytenucleoside be photoactivated by exposure to UV light, again resulting in transporters are band 4.5 polypeptides, a conclusion selective labeling of the band 4.5 region of SDS-polyacrylsupported by previous isolation studies based on the amide gels
As was the case with protein-depleted membranes, irradiation of ghosts containing site-bound [3HjNBMPR with high intensity UV light resulted in substantial covalent incorporation of radioactivity
Summary
Pig, and sheep blood was collected into heparin and the erythrocytes washed three times with a medium containing 140 mM NaCI, 5 mM KC1, 20 mM Tris-HC1 (pH 7.4 at 22 "C), 2 mM MgCl,, and 0.1 mM EDTA (disodium salt).The buffy coat was discarded. Hemoglobin-free erythrocyte membranes ("ghosts"), prepared by osmotic lysis as described by Dodge et al [27], were resuspended in 5 mM sodium phosphate (pH 8.0 at 22 " C )and stored at -70 "C for up to 1 month. "Protein-depleted" membranes were prepared using 0.1 mM EDTA (pH 11.2) as previously described [9] and resuspended in phosphate medium
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