Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung

Shi, Maggie M.; Wu, Jin-Shyun Ruth; Lee, Chi-Ming; Young, James D.

doi:10.1016/0006-291x(84)91344-5

Cited by 43 publications

(8 citation statements)

References 12 publications

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“…The inability of [ 3 H]NBMPR to photolabel mENT1⌬11 suggests that the loss of this C-terminal region of mENT1 removes the residue that NBMPR normally cross-links to or that a critical residue has shifted (Kwong et al, 1993), we would argue that the loss of the last three transmembrane domains of mENT1 leads to a conformation change that prevents covalent attachment of the [ 3 H]N-BMPR to elements of its binding pocket in the N-terminal part of the protein. It is generally believed that the S-nitrobenzyl group of NBMPR is photoactivated upon exposure to UV light; hence the amino acid residue involved in the photoaffinity labeling is probably proximal to the mENT1 region that binds the S-nitrobenzyl moiety of NBMPR (Paterson and Oliver, 1971;Young et al, 1983;Shi et al, 1984;Zhu et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Robillard

Bone²,

Park³

et al. 2008

Mol Pharmacol

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Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1⌬11) missing the last three transmembrane domains of the full-length mENT1. The mENT1⌬11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1⌬11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1⌬11 in terms of transport function or inhibitor binding affinity. PK15-mENT1⌬11 transfected cells bound the ENT1 3 H]uridine. The only significant differences between the mENT1 variants were that mENT1⌬11 could not be photolabeled with [3 H]NBMPR and that mENT1⌬11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation.

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Section: Discussionmentioning

confidence: 99%

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Robillard

Bone²,

Park³

et al. 2008

Mol Pharmacol

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“…In contrast to human erythrocytes where the potency for inhibition of NBMPR binding by NBTGR, dilazep and dipyridamole differ by less than 10-fold (Hammond & Cianachan, 1984), the IC50 values for inhibition for binding to rat erythrocyte membranes differed by 4 orders of magnitude (0.08, 12 and 1000 nM for NBTGR, dilazep and dipyridamole, respectively). The lower potency of dilazep and dipyridamole for inhibition of [3H]NBMPR binding has also been observed for cortical, liver, cardiac muscle and lung rat membranes when compared to the membranes prepared from guinea pig tissues (Hammond & Clanachan, 1984;Shi et al, 1984;Williams et al, 1984;Verma & Marangos, 1985). The NBMPR-sensitive nucleoside transporter in human and fetal sheep erythrocytes has been demonstrated to be chemically asymmetric with respect to pCMBS inhibition of transport activity, inhibition only occurring when the organomercurial has access to the cytoplasmic membrane surface (Jarvis & Young, 1982;Tse et al, 1985).…”

Section: ~I0mentioning

confidence: 90%

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate

Jarvis

Young

1986

J. Membrain Biol.

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The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial, p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 microM) by NBMPR was biphasic--35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 microM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparent Km for uridine 50 +/- 18 and 163 +/- 28 microM, respectively). The two transport systems also differed in their sensitivity to pCMBS. NBMPR-insensitive uridine transport was inhibited by pCMBS with an IC50 of approximately 25 microM, while 1 mM pCMBS had little effect on NBMPR-sensitive transport by intact cells. pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by beta-mercaptoethanol, suggesting that the pCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparent Kd 46 +/- 25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25 +/- 6 molecules/site per sec at 22 degrees C. pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR and pCMBS.

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“…[ (Wong et al, 1983;Shi et al, 1984;Hon et al, 1990b (Figure 3). This effect seemed to be specific for sodium because potassium chloride tested between 1.5 and 150mM did not significantly affect [3H]-PAF binding ( Figure 3).…”

Section: Preparation Ofplateletsmentioning

confidence: 99%

“…Specific binding was calculated by subtracting nonspecific binding from total binding. Other radioligand binding assays were performed as described previously (Wong et al, 1983;Shi et al, 1984;Hon et al, 1990b). The binding of [3H]-nitrendipine to dihydropyridine-sensitive calcium channel was determined with rat cerebral cortex membranes.…”

Section: Preparation Ofplateletsmentioning

confidence: 99%

Prehispanolone, a novel platelet activating factor receptor antagonist from Leonurus heterophyllus

Lee

Jiang

Shang

et al. 1991

British J Pharmacology

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Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung

Cited by 43 publications

References 12 publications

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate

Prehispanolone, a novel platelet activating factor receptor antagonist from Leonurus heterophyllus

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