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

1984 ◽  
Vol 62 (12) ◽  
pp. 1283-1287
Author(s):  
Alan R. P. Paterson ◽  
Anthony F. Almeida ◽  
Ewa Dahlig-Harley ◽  
Jon P. Miller ◽  
Carol E. Cass
Keyword(s):  
Uv Light ◽  
Covalent Binding ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Covalent Attachment ◽  
Erythrocyte Membranes ◽  
Gel Chromatography ◽  
Intact Cells ◽  
High Affinity ◽  
Covalently Linked

The potent inhibitor of nucleoside transport, 6-[(4-nitrobenzyl)mercapto]-9-β-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 °C through 9 × 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.

Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes

1988 ◽  
Vol 66 (8) ◽  
pp. 839-852 ◽  
Author(s):  
James D. Craik ◽  
A. Heather Good ◽  
Russell Gottschalk ◽  
Simon M. Jarvis ◽  
Alan R. P. Paterson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cytochalasin B ◽  
Cultured Cells ◽  
Uv Light ◽  
Binding Protein ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Intact Cells ◽  
Neonatal Pigs ◽  
Limited Digestion

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55 000 and 64 000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44 000 and 57 000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18 000 – 23 000 and 43 000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.

scholarly journals Nucleoside transport in human and sheep erythrocytes. Evidence that nitrobenzylthioinosine binds specifically to functional nucleoside-transport sites

1980 ◽  
Vol 190 (2) ◽  
pp. 377-383 ◽  
Author(s):  
S M Jarvis ◽  
J D Young
Keyword(s):  
Transport System ◽  
Binding Sites ◽  
Sheep Erythrocyte ◽  
Specific Interaction ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Sheep Erythrocytes ◽  
High Affinity ◽  
Transport Site

Nitrobenzyl[35S]thioinosine binding and nitro[3H]benzylthioinosine binding to nucleoside-permeable and nucleoside-impermeable sheep erythrocyte membranes was investigated, and compared with that found for human erythrocytes. High-affinity nitrobenzylthioinosine-binding sites (apparent KD congruent to 1 nM) were present on human and nucleoside-permeable but not nucleoside-impermeable sheep erythrocyte membranes (8400 and 18 sites/cell for human and sheep nucleoside-permeable sheep erythrocytes was displaced by nitrobenzylthioguanosine and dipyridamole. Uridine, inosine and adenosine inhibited binding. The smaller number of nitrobenzylthioinosine sites on nucleoside-permeable cells compared with human erythrocytes corresponded to a considerably lower Vmax. for uridine influx in these cells (0.53 × 10(-20) mol/cell per s at 25 degrees C compared with 254 × 10(-20) mol/cell per s). It is suggested that high-affinity nitrobenzylthioinosine binding represents a specific interaction with functional nucleoside-transport sites. The uridine-translocation capacity for each transport site at 25 degrees C is 180 molecules/site per s for both nucleoside-permeable sheep cells and human erythrocytes (assuming a 1:1 interaction between nitrobenzylthioinosine and the nucleoside-transport system).

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

1988 ◽  
Vol 66 (10) ◽  
pp. 1126-1133 ◽  
Author(s):  
Elena Burdett ◽  
Amira Klip
Keyword(s):  
Metabolic Regulation ◽  
Glucose Transporter ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Glucose Transporters ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Western Blots ◽  
Intact Cells ◽  
Human Erythrocyte Membranes

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55 000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze–thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters. It is concluded that the antibody may be reacting with exofacial regions of the glucose transporter in intact cells. This suggests that the antibodies may be used to detect relative changes in glucose transporter number on the cell surface.

scholarly journals Reconstitution studies of amino acid transport system L in rat erythrocytes

1993 ◽  
Vol 292 (3) ◽  
pp. 655-660 ◽  
Author(s):  
S Y M Yao ◽  
R George ◽  
J D Young
Keyword(s):  
Amino Acid ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Phospholipid Vesicles ◽  
Transport Activity ◽  
Protein Ratio ◽  
Intact Cells ◽  
Amino Acid Transport System ◽  
Simple Diffusion ◽  
System L

In many cell types, including human erythrocytes, membrane transport of hydrophobic amino acids such as leucine and phenylalanine is mediated primarily by Na(+)-independent system L. In this paper we demonstrate that erythrocytes from the rat have a 400-fold higher system L transport capacity than human erythrocytes. We have exploited this high transport activity to achieve the first successful reconstitution of an erythrocyte amino acid transporter into phospholipid vesicles. Rat erythrocyte membranes were depleted of extrinsic membrane proteins, solubilized in 50 mM n-octyl glucoside and reconstituted into egg-yolk phospholipid vesicles by a gel filtration freeze-thaw protocol. Optimal reconstitution of transport activity occurred at lipid/protein ratios of 25-35:1. At a lipid/protein ratio of 25:1, one-half of the total uptake of L-[14C]leucine (0.2 mM, 25 degrees C) was inhibited by 2 mM phloretin and thus judged to be carrier-mediated. This component of L-leucine uptake was inhibited by non-radioactive L-phenylalanine and L-leucine, and only to a very much weaker extent by glycine and L-alanine. Two other inhibitors of system L in intact cells, MK196 and PCMBS (p-chloromercuriphenylsulphonate), were also effective inhibitors of phloretin-sensitive L-leucine transport in reconstituted proteoliposomes. Phloretin-insensitive uptake of L-leucine in proteoliposomes occurred by simple diffusion across the lipid bilayer.

Binding of the Nucleoside Transport Inhibitor 4-Nitrobenzylthioinosine to Erythrocyte Membranes

1973 ◽  
Vol 51 (5) ◽  
pp. 666-672 ◽  
Author(s):  
M. A. Pickard ◽  
R. R. Brown ◽  
B. Paul ◽  
A. R. P. Paterson
Keyword(s):  
Binding Sites ◽  
Nucleoside Transport ◽  
Erythrocyte Membranes ◽  
Affinity Binding ◽  
Inhibitor Molecule ◽  
High Affinity Binding ◽  
High Affinity ◽  
Transport Inhibitor ◽  
Binding Data ◽  
Nucleoside Transport Inhibitor

4-Nitrobenzylthioinosine (NBMPR), a potent nucleoside transport inhibitor, was prepared in two radioactive forms and the binding of these to erythrocyte ghosts was studied. Similar binding data were obtained with inhibitor containing 14C in the purine 8-position or in the benzyl 7-position, suggesting that the entire inhibitor molecule was bound. A saturable high-affinity mode of NBMPR binding was apparent; NBMPR bound in this way was not removed by washing, but was displaced by a related inhibitor of nucleoside transport, 2-hydroxy-5-nitrobenzylthioguanosine (HNBTGR). It is postulated that the high-affinity binding sites are the nucleoside transport elements of the erythrocyte membrane. From ghosts treated with 14C-NBMPR under conditions which assured binding of the high affinity type, 14C was recovered by extractions in the form of NBMPR. Thus, this mode of NBMPR binding is reversible and covalent linkages do not appear to be involved. A low affinity mode of NBMPR binding was also demonstrated; this appeared to be a partition of NBMPR between the medium and the membrane substance. This component of bound NBMPR was not displaced by HNBTGR and was removed by washing.

scholarly journals The low C5 convertase activity of the C4A6 allotype of human complement component C4

1989 ◽  
Vol 261 (3) ◽  
pp. 743-748 ◽  
Author(s):  
T Kinoshita ◽  
A W Dodds ◽  
S K A Law ◽  
K Inoue
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Alternative Pathway ◽  
Covalent Attachment ◽  
Haemolytic Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Covalently Linked

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system

1985 ◽  
Vol 63 (10) ◽  
pp. 1302-1307 ◽  
Author(s):  
James R. Hammond ◽  
Evan F. Williams ◽  
Alexander S. Clanachan
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Transport System ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Rat Tissues ◽  
High Affinity ◽  
Cardiac Membranes ◽  
Transport Inhibitors ◽  
Calcium Channel Inhibitors

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (<50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (> 1 μM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (> 1 μM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.

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

1983 ◽  
Vol 216 (2) ◽  
pp. 299-308 ◽  
Author(s):  
R Koren ◽  
C E Cass ◽  
A R Paterson
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Animal Cells ◽  
Equilibrium Conditions ◽  
Intact Cells ◽  
High Affinity

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.

scholarly journals Inhibition by nucleosides of glucose-transport activity in human erythrocytes

1988 ◽  
Vol 249 (2) ◽  
pp. 383-389 ◽  
Author(s):  
S M Jarvis
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Binding Activity ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Transport Activity ◽  
Intact Cells ◽  
Order Of Magnitude ◽  
Glucose Carrier

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.

scholarly journals Non-Covalent Binding of Tripeptides-Containing Tryptophan to Polynucleotides and Photochemical Deamination of Modified Tyrosine to Quinone Methide Leading to Covalent Attachment

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4315
Author(s):  
Antonija Erben ◽  
Igor Sviben ◽  
Branka Mihaljević ◽  
Ivo Piantanida ◽  
Nikola Basarić
Keyword(s):  
Quantum Yield ◽  
Photophysical Properties ◽  
Covalent Binding ◽  
Quinone Methide ◽  
Covalent Attachment ◽  
Dna And Rna ◽  
Pair Composition ◽  
Aqueous Solvent ◽  
Fluorometric Study ◽  
Spectral Responses

A series of tripeptides TrpTrpPhe (1), TrpTrpTyr (2), and TrpTrpTyr[CH2N(CH3)2] (3) were synthesized, and their photophysical properties and non-covalent binding to polynucleotides were investigated. Fluorescent Trp residues (quantum yield in aqueous solvent ΦF = 0.03–0.06), allowed for the fluorometric study of non-covalent binding to DNA and RNA. Moreover, high and similar affinities of 2×HCl and 3×HCl to all studied double stranded (ds)-polynucleotides were found (logKa = 6.0–6.8). However, the fluorescence spectral responses were strongly dependent on base pair composition: the GC-containing polynucleotides efficiently quenched Trp emission, at variance to AT- or AU-polynucleotides, which induced bisignate response. Namely, addition of AT(U) polynucleotides at excess over studied peptide induced the quenching (attributed to aggregation in the grooves of polynucleotides), whereas at excess of DNA/RNA over peptide the fluorescence increase of Trp was observed. The thermal denaturation and circular dichroism (CD) experiments supported peptides binding within the grooves of polynucleotides. The photogenerated quinone methide (QM) reacts with nucleophiles giving adducts, as demonstrated by the photomethanolysis (quantum yield ΦR = 0.11–0.13). Furthermore, we have demonstrated photoalkylation of AT oligonucleotides by QM, at variance to previous reports describing the highest reactivity of QMs with the GC reach regions of polynucleotides. Our investigations show a proof of principle that QM precursor can be imbedded into a peptide and used as a photochemical switch to enable alkylation of polynucleotides, enabling further applications in chemistry and biology.

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