Erythrocyte nucleoside and sugar transport Endo-<i>β</i>-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins

Kwong, F. Y. P.; Baldwin, Stephen A.; Scudder, Peter; Jarvis, Simon M.; Choy, M. Y. M.; Young, James D.

doi:10.1042/bj2400349

Cited by 46 publications

(6 citation statements)

References 30 publications

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“…Trypsin digestion (10/xg per mg protein for 15 rain at 22~ resulted in complete cleavage of the 45,000-77,000 radiolabeled protein, but in contrast to human erythrocytes no discrete smaller M r fragments were resolved on the SDS-gel electropherogram [13]. Figure 8 shows that endoglycosidase F cleavage caused a shift of the 3H peak to a lower apparent M r of 46,000, a value similar to that previously obtained with the endoglycosidase F treated human erythrocyte nucleoside transporter [24]. The electrophoretic mobility of the presumed degradation 3H peak was also shifted to a lower-M r region of the gel, suggesting the polypeptide fragment(s) are also glycosylated.…”

Section: H]nbmpr Binding To Brush-border and Basal Membranessupporting

confidence: 65%

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Barros

et al. 1991

J. Membrain Biol.

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The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2'-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2'-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparent Km approx. 150 microM) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparent Kd 0.98 +/- 0.21 nM). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparent Ki values were 0.23 +/- 0.012, 0.36 +/- 0.035, 0.78 +/- 0.1, 0.70 +/- 0.12 (mM), and 0.12 and 4.2 +/- 1.4 (nM). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brush-border membrane vesicles (apparent Kd 1.05 +/- 0.13 nM and apparent Ki values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14 +/- 0.045, 0.54 +/- 0.046, 1.26 +/- 0.20, 1.09 +/- 0.18 mM and 0.14 and 3.7 +/- 0.5 nM, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparent Mr on SDS gel electropherograms of 77,000-45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surfaces of the human placental syncytiotrophoblast possess broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.

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Section: H]nbmpr Binding To Brush-border and Basal Membranessupporting

confidence: 65%

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Barros

et al. 1991

J. Membrain Biol.

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“…Previous studies have shown that both cloned and endogenous ENT1 are glycoproteins (24,25). However, there is no experimental evidence to support that hENT2 is a glycoprotein.…”

Section: Generation Of a Nucleoside Transporter-deficient Pk15ntdmentioning

confidence: 84%

Kinetic and Pharmacological Properties of Cloned Human Equilibrative Nucleoside Transporters, ENT1 and ENT2, Stably Expressed in Nucleoside Transporter-deficient PK15 Cells

Ward

Sherali

et al. 2000

Journal of Biological Chemistry

282

297

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We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC 50 , 0.4 ؎ 0.1 nM versus 2.8 ؎ 0.3 M) and dipyridamole (IC 50 , 5.0 ؎ 0.9 nM versus 356 ؎ 13 nM), respectively. [ 3 H]NBMPR binds to ENT1 cells with a high affinity K d of 0.377 ؎ 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7.7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [ 3 H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.

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“…In view of these observations, the reltttionship of the NBMPR binding site to the permeant site remains uncertain. It should also be added that photoaffinity labeling of the plasma membrane of human erythrocytes with 3H-NBMPR has facilitated the partial purification of the binding protein [97][98][99][100][101][102][103][104].…”

Section: Kinetic and Physiological Propertiesmentioning

confidence: 99%

Membrane transport and the antineoplastic action of nucleoside analogues

Sirotnak

Barrueco

1987

Cancer Metast Rev

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This article summarizes recent studies characterizing nucleoside transport in mammalian cells and discusses evidence for a role of membrane transport in the pharmacologic action of nucleoside analogues. Some of these studies have also addressed the controversy concerning the multiplicity in transport routes. It seems clear that erythrocytes and, perhaps, some other mammalian cells possess a single, broadly specific system for transporting nucleosides. However, substantial evidence from valid studies discriminating between transport and intracellular metabolism suggests that at least some mammalian cells, including some tumor cells, possess more than a single system. Evidence now exists for a determining role of membrane transport of nucleoside analogues in their cytotoxicity and, in the case of one pyrimidine nucleoside (AraC), in therapeutic responsiveness in leukemic patients. There are also numerous examples of transport-related resistance to nucleoside analogues. Included in this article are the results of studies from the authors' laboratory pertaining to the therapeutic activity of the purine nucleoside, FAraA, in murine tumor models. These studies provide evidence for a determining role of both membrane transport and intracellular phosphorylation in the selective antitumor action of this agent against murine leukemia. Substantially increased transport inward of FAraA occurs at pharmacologically achievable concentrations of this agent in tumor cells as compared to drug-limiting, normal proliferative epithelium of the small intestine. The basis for this differential appears to be the kinetic duality of FAraA and adenosine transport inward found in tumor cells, but not in proliferative intestinal epithelial cells. Tumor cells have highly saturable (low influx Km) and poorly saturable (high influx Km) systems for adenosine transport, both of which are shared by FAraA. In contrast, proliferative epithelial cells have only a poorly saturable system for these substrates. If a similar kinetic duality of nucleoside transport is found in other tumor cells certain implications arise concerning the significance of the duality to neoplastic transformation.

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Erythrocyte nucleoside and sugar transport Endo-β-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins

Cited by 46 publications

References 30 publications

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Kinetic and Pharmacological Properties of Cloned Human Equilibrative Nucleoside Transporters, ENT1 and ENT2, Stably Expressed in Nucleoside Transporter-deficient PK15 Cells

Membrane transport and the antineoplastic action of nucleoside analogues

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