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Extraction and partial purification of the nucleoside-transport system from human erythrocytes based on the assay of nitrobenzylthioinosine-binding activity.

ABSTRACT: Nitrobenzylthioinosine, a potent nucleoside-transport inhibitor, binds to high-affinity sites on the human erythrocyte membrane. This binding is a specific interaction with functional nucleoside-transport sites. The protein(s) responsible for high-affinity nitrobenzylthioinosine binding was purified 13-fold by treatment of haemoglobin-free 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with Triton X-100 and passage of the soluble extract through a DEAE-cellulose column equilibrated with Triton X-100. Void-volume fractions were collected and treated with Bio-Beads SM-2 to remove detergent. These fractions contained 31% of the starting nitrobenzylthioinosine-binding activity. They also contained D-glucose-sensitive cytochalasin B-binding activity. Nitrobenzylthioinosine binding to the partially purified preparation was saturable (apparent Kd 1.6 nM) and inhibited by nitrobenzylthioguanosine, dipyridamole and uridine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pooled void-volume fractions revealed the presence of only two detectable protein bands, the broad zone 4.5 (containing glucose-transport protein) and a small amount of band 7.

SUBMITTER: Jarvis SM

PROVIDER: S-EPMC1162748 | biostudies-other | 1981 Jan

REPOSITORIES: biostudies-other

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Project description:We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.

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Extraction and partial purification of the nucleoside-transport system from human erythrocytes based on the assay of nitrobenzylthioinosine-binding activity.

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