Journal ArticleDOI
Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate
Simon M. Jarvis,James D. Young +1 more
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TLDR
It is concluded that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR and pCMBS.Abstract:
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 μM) 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 μM. 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 (apparentKm for uridine 50±18 and 163±28 μM, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC50 of ∼25μM, while 1 mMpCMBS 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 β-mercaptoethanol, suggesting that thepCMBS-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 (apparentKd46±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°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 andpCMBS.read more
Citations
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Nucleoside and nucleobase transport systems of mammalian cells
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Nucleoside and nucleobase transport in animal cells
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Adenosine receptors: expression, function and regulation.
TL;DR: The biochemical characteristics and signaling cascade associated with each receptor are described and insight is provided into how these receptors are regulated in response to agonists are provided.
Journal ArticleDOI
Molecular Cloning and Functional Characterization of Nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) Equilibrative Nucleoside Transporter Proteins (rENT1 and rENT2) from Rat Tissues
Sylvia Y.M. Yao,Amy M. L. Ng,William R. Muzyka,Mark Griffiths,Mark Griffiths,Carol E. Cass,Stephen A. Baldwin,Stephen A. Baldwin,James D. Young +8 more
TL;DR: Observations demonstrate thates and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.
Journal ArticleDOI
Recent advances in the molecular biology of nucleoside transporters of mammalian cells
TL;DR: From the sequence relationships of these proteins with each other and with sequences in the public data bases, it is concluded that the equilibrative and concentrative nucleoside transport processes are the same.
References
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Journal ArticleDOI
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
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Cleavage of structural proteins during the assemble of the head of bacterio-phage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Journal ArticleDOI
The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes
TL;DR: The effects of the ionic strength and pH of the hemolyzing solution on the hemoglobin content of human erythrocyte ghosts were studied in phosphate buffers and suggest an electrophysical interaction of hemoglobin with membrane constituents.
Journal ArticleDOI
The removal of leukocytes and platelets from whole blood.
TL;DR: This procedure is rapid, reliable, removes over 99.75 per cent of the leukocytes from blood, and does not seem selectively to retain reticulocytes or to release a significant proportion of leukocyte enzymes.