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A R Paterson

Publications and source records attributed to A R Paterson.

At least 55 records · Page 3Linked to original sources
PubMed1986

Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells.

Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.

Adenosine↗
PubMed1986

Nitrobenzylthioinosine: an in vivo inhibitor of pig erythrocyte energy metabolism.

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.

Adenosine Diphosphate↗
PubMed1985

Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate.

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.

Adenosine↗
PubMed1985

Reconstitution studies of the human erythrocyte nucleoside transporter.

The human erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide (Mr 45,000-66,000) on the basis of reversible binding and photoaffinity labeling experiments with the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). In the present study, the NBMPR-binding protein was extracted from protein-depleted human erythrocyte "ghosts" with Triton X-100 and reconstituted into soybean phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes exhibited nitrobenzylthioguanosine (NBTGR)-sensitive [14C]uridine transport. A partially purified preparation of the NBMPR-binding protein, consisting largely of band 4.5 polypeptides, was also shown to have nucleoside transport activity. This band 4.5 preparation exhibited a 10-fold increase in uridine transport activity and a 7-fold increase in NBMPR-binding activity relative to the crude membrane extract. Uridine transport by the reconstituted band 4.5 preparation was saturable (apparent Km = 0.21 mM; Vmax = 9 nmol/mg of protein/5 s) and was inhibited by dipyridamole, dilazep, adenosine, and inosine. The vesicles reconstituted with the band 4.5 preparation also exhibited stereospecific glucose transport which was inhibited by cytochalasin B, but unaffected by NBTGR. In contrast, cytochalasin B was a poor inhibitor of NBTGR-sensitive uridine transport. These experiments implicate band 4.5 polypeptides in both nucleoside and sugar permeation.

Biological Transport, Active↗
PubMed1985

Toxicologic and antitumor studies on 5-hydroxymethyldeoxyuridine.

Toxic effects of 5-hydroxymethyldeoxyuridine (HMUdR) were studied in white Swiss mice. Pathological, hematological, and clinical chemistry parameters were examined. Systemic toxicity was not observed in mice after ip administration of HMUdR in single doses up to 2000 mg/kg. Mice administered HMUdR daily for 15 days at 200 mg/kg, ip, manifested loss of weight, rough hair coat, diarrhea, swollen abdomens, weakness, lethargy, and a 20% mortality rate. Hematological and clinical chemistry parameters of all mice receiving HMUdR were within normal limits. At necropsy, all organs were grossly normal but microscopic examination of tissues of treated mice revealed the presence of shortened, thickened villi in the small intestine, nuclear vacuolation and necrosis of intestinal crypt epithelial cells, and some cytoplasmic vacuolation causing nuclear margination in hepatocytes. All histological lesions were reversible with cessation of treatment. HMUdR was active against murine L1210 and L5178Y leukemias in cell culture. The concentrations required to inhibit cell growth 50% compared to untreated cells was 2 and 4 microM, respectively. When HMUdR was administered ip at 50, 100, or 200 mg/kg in five daily doses to mice implanted with L1210 cells, life spans increased 20, 30, or 33% over placebo-treated controls.

Animals↗
PubMed1985

The effect of pH on interaction of nitrobenzylthioinosine and hydroxynitrobenzylthioinosine with the nucleoside transporter of human erythrocyte membranes.

Site-specific binding to human erythrocyte membranes of nitrobenzylthioinosine (NBMPR), un-ionized at physiological pH, was compared with that of hydroxynitrobenzylthioinosine (HNBMPR), pKa 6.4, at graded pH values. Binding of [3H]NBMPR was measured directly, and that of HNBMPR was assayed by competitive inhibition by HNBMPR of [3H]NBMPR binding. Kd and Bmax values for binding of [3H]NBMPR to erythrocyte membranes were independent of pH. Kd values for the competing ligand were determined by mass law analysis of equilibrium binding data using either (a) apparent ligand concentration (dissociated plus undissociated forms of HNBMPR) or (b) the concentration of undissociated HNBMPR. Kd values for HNBMPR calculated with the apparent ligand concentration increased 10-fold as the fraction of HNBMPR molecules present in the dissociated form was increased (by pH changes) from 14 to 88%, whereas Kd values for the undissociated form of HNBMPR were independent of pH. The results presented here demonstrate that the undissociated form of HNBMPR binds more tightly to the transport-inhibitory sites of erythrocytes than NBMPR and suggest that ionization of S6-substituted thiopurine ribonucleosides eliminates or greatly decreases their ability to interact with the binding sites.

Affinity Labels↗
PubMed1984

Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method.

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.

Adenosine↗
PubMed1984

Dephosphorylation of nitrobenzylthioinosine 5'-monophosphate by ecto 5'-nucleotidase of HeLa cells.

HeLa cells as well as human and mouse erythrocytes possess membrane sites which bind the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), reversibly but tightly (KD, 10(-9)-10(-10) M). Site-specific binding of the ligand correlates with inhibition of nucleoside transport. The present study showed that the 5'-phosphate of NBMPR, NBMPR-P, was not transport inhibitory. Upon exposure to [35S]NBMPR-P or [G-3H]NBMPR-P, HeLa cells retained the isotopic labels virtually exclusively in the form of NBMPR. The dephosphorylation of [G-3H]NBMPR-P by HeLa cells, assayed by the production of extracellular [G-3H]NBMPR, was competitively inhibited by AMP, but was not affected by the presence of 5 microM NBMPR, a concentration sufficient to completely occupy the transport inhibitory sites. Thus, the sites at which dephosphorylation of NBMPR occurs in HeLa cells are separate from and function independently of the high affinity sites which bind NBMPR.

5'-Nucleotidase↗
PubMed1984

Photoaffinity labelling of the nucleoside transporter of cultured mouse lymphoma cells.

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [3H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that 3H was present in polypeptides which migrated as a major band with an apparent Mr of 45000-65000.

Affinity Labels↗
PubMed1984

Identification of the nucleoside transporter in cultured mouse lymphoma cells. Photoaffinity labeling of plasma membrane-enriched fractions from nucleoside transport-competent (S49) and nucleoside transport-deficient (AE1) cells with [3H]nitrobenzylthioinosine.

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.

Adenosine↗
PubMed1984

Nucleoside transport in human erythrocytes. Nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes.

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Biological Transport↗
PubMed1984

Separation of nitrobenzylthioinosine from high affinity binding sites on human erythrocytes by gel chromatography.

The potent inhibitor of nucleoside transport, 6-[(4-nitrobenzyl) mercapto]-9-beta-D-ribofuranosylpurine (NBMPR), binds reversibly, but with high affinity (KD 0.3-1.4 nM) to plasma membrane sites on human erythrocytes, and occupancy of those sites by NBMPR correlates with inhibition of transport. An earlier study from this laboratory showed that, upon photoactivation of site-bound [3H]NBMPR on erythrocyte membranes by exposure to UV light, isotopic ligand molecules became covalently linked to membrane polypeptides, which migrated as a discrete band on gel electrophoretograms, thereby identifying nucleoside transport polypeptides. The present study showed that erythrocytes on which the high affinity sites were occupied by reversibly bound [3H]NBMPR were freed of the latter by slow passage at 37 degrees C through 9 X 300 mm columns of Sephadex G-200 gel. Photoactivation of site-bound [3H]NBMPR on the intact cells caused apparent covalent attachment of the ligand because cells so treated retained the isotopic label during passage through the gel columns. Apparent covalent binding to erythrocytes also resulted from photoactivation of site-bound [3H]nitrobenzylmercaptopurine arabinoside.

Chromatography, Gel↗
PubMed1984

Transport of uridine and 3-deazauridine in cultured human lymphoblastoid cells.

The transport of uridine and 3-deazauridine was compared in two lines of cultured human lymphoblastoid cells that differ in their sensitivity to 3-deazauridine apparently because of reduced uridine-cytidine kinase activity in the resistant line. The kinetic parameters (+/- S.E.) of uridine transport were similar in the two cell lines: Km, 0.23 +/- 0.02 and 0.25 +/- 0.07 mM; and Vmax, 35 +/- 2 and 57 +/- 10 pmol/microliter of cell water per sec, respectively, for 6410/MP (parental) and 6410/MP/DU (resistant) cells. 3-Deazauridine, while transported with similar kinetic characteristics in both cell lines, was not as good a substrate for the nucleoside transporter as was uridine, and its transport was dependent on pH. Kinetic parameters, determined using calculated concentrations of the undissociated form of 3-deazauridine (pKa, 6.5), were: Km, 0.52 +/- 0.01 and 0.51 +/- 0.03 mM; and Vmax, 28 +/- 0.5 and 24 +/- 0.9 pmol/microliter of cell water per sec, respectively, for 6410/MP and 6410/MP/DU cells. At pH 8, a condition in which 97% of 3-deazauridine molecules are ionized, rates of transport were almost zero. It is concluded that the undissociated form of 3-deazauridine is the substrate for the nucleoside transporter.

3-Deazauridine↗
PubMed1983

The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells.

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.

Animals↗
PubMed1983

Benzodiazepine inhibition of nucleoside transport in human erythrocytes.

The interaction of several benzodiazepines (BDZs) with the nucleoside transport system of fresh erythrocytes from humans was investigated. The affinities of BDZs for the nucleoside transport system were estimated by measuring BDZ inhibition of (a) the site-specific binding of nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport, and (b) the uridine transport processes, zero-trans influx, zero-trans efflux, and equilibrium exchange influx. The BDZs inhibited both the inward and outward transport processes, and, for individual agents, inhibition constants (Ki) were similar for the inhibition of each transport process and for the inhibition of the site-specific binding of nitrobenzylthioinosine. The order of potencies of the BDZs in their interactions with the nucleoside transport mechanism (Ro 5-4864 greater than diazepam greater than clonazepam greater than lorazepam greater than flurazepam) is distinct from the potencies of these compounds at BDZ recognition sites. The affinities of the BDZs for the nucleoside transport system, which are about 1000-fold lower than for BDZ recognition sites, suggest that significant inhibition is unlikely to occur with the plasma concentrations (less than 1 microM) that result from usual anxiolytic doses of these agents.

Benzodiazepines↗
PubMed1983

Photoaffinity labeling of the human erythrocyte nucleoside transporter by N6-(p-Azidobenzyl)adenosine and nitrobenzylthioinosine. Evidence that the transporter is a band 4.5 polypeptide.

N6-(p-Azidobenzyl)adenosine (ABA) and nitrobenzylthioinosine (NBMPR) were employed as covalent probes of the nucleoside transport mechanism in human erythrocytes. NBMPR, a potent inhibitor of nucleoside transport, binds tightly (KD 0.3-1 nM) to specific sites on nucleoside transporter elements. ABA, a less potent inhibitor of uridine influx, competitively inhibited NBMPR binding (Ki 15 nM). [3H]ABA was bound tightly (KD 13.4 nM) but reversibly to sites on erythrocytes which appeared to be those which bind NBMPR. ABA binding was inhibited by uridine and adenosine. Irradiation with UV light caused site-bound [3H]ABA on erythrocyte membranes to become covalently bound and, similarly, photoactivation resulted in covalent attachment of membrane-bound [3H]NBMPR. In the presence of dithiothreitol, a free radical scavenger, photoactivation of the site-bound 3H-ligand on membranes depleted of extrinsic membrane proteins resulted in selective incorporation of 3H into band 4.5 of the membrane polypeptides which were resolved on sodium dodecyl sulfate-polyacrylamide gel electropherograms. This result, when considered with previous findings, indicates that the NBMPR-binding component of the nucleoside transport mechanism (or the entire mechanism, if the NBMPR site is an integral part) is a band 4.5 polypeptide.

Affinity Labels↗
PubMed1983

Nucleoside transport in human erythrocytes. A simple carrier with directional symmetry in fresh cells, but with directional asymmetry in cells from outdated blood.

Kinetic characteristics of the transport of uridine, a non-metabolized permeant in human erythrocytes, have been compared in erythrocytes from fresh and outdated stored blood. Uridine transport kinetics in fresh cells conformed to the predictions of a simple carrier model operating with directional symmetry, but in erythrocytes from outdated blood the kinetic characteristics of uridine transport were those of an asymmetric system. The latter result agrees with earlier reports by others. The mobility of the loaded and empty carriers differed by about 6- and 12-fold in fresh and outdated blood, respectively.

Binding Sites↗