Nucleoside Transport. I. A Mediated Process in Human Erythrocytes

1971 ◽  
Vol 49 (2) ◽  
pp. 262-270 ◽  
Author(s):  
J. M. Oliver ◽  
A. R. P. Paterson
Keyword(s):  
Competitive Inhibition ◽  
Diffusion Mechanism ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Uridine Uptake ◽  
Saturation Kinetics ◽  
Pyrimidine Bases ◽  
Thymidine Transport ◽  
Broad Specificity

The transport of nucleosides by human erythrocytes has been studied by measuring disappearance of 14C-labelled uridine and thymidine from incubation media; saturation kinetics and competitive inhibition of uridine uptake by formycin B were observed. Uridine and thymidine transport were nonconcentrative under incubation conditions in which cellular ATP concentrations were maintained. Efflux of uridine or thymidine from preloaded cells was induced by an inward flow of various nucleosides, but not by purine or pyrimidine bases. It is concluded that the transport of nucleosides across the plasma membrane of human erythrocytes is accomplished by a nonconcentrative, 'facilitated diffusion' mechanism of broad specificity.

scholarly journals The effects of oestradiol on nucleoside transport in rat uterus

1971 ◽  
Vol 121 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Janet M. Oliver
Keyword(s):  
Cytosine Arabinoside ◽  
Concentration Gradient ◽  
Initial Rate ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Rat Uterus ◽  
Intracellular Space ◽  
Uridine Uptake

1. By using the non-metabolized cytidine analogue, cytosine arabinoside, it was possible to examine the mechanism of nucleoside transport in the immature rat uterus in the absence of intracellular utilization of the permeant. It was demonstrated that the uptake of cytosine arabinoside is not accumulative and that it can be competitively inhibited by the addition of a second nucleoside, uridine. Introduction of a concentration gradient of uridine from the medium towards the intracellular water promotes the counterflow of cytosine arabinoside out of the cells against its concentration gradient. These properties indicate that a facilitated-diffusion system is involved in nucleoside transport in the uterus. Further counterflow studies have shown that the transport system has a broad specificity for purine and pyrimidine nucleosides and that it is distinct from the processes that mediate the uptake of sugars, amino acids and purine and pyrimidine bases. 2. Oestradiol injection has no effect on the initial rate of cytosine arabinoside uptake in vitro. The increased amount of the analogue taken up per uterus is simply due to the expansion of the uterine volume that accompanies oestrogen action. 3. It is concluded that the striking increase in uridine uptake, observed in vivo in uteri from oestrogen-treated rats, does not result from an increase in the initial rate of nucleoside transport into the intracellular space of the tissue.

Nucleoside Transport. II. Inhibition by p-Nitrobenzylthioguanosine and Related Compounds

1971 ◽  
Vol 49 (2) ◽  
pp. 271-274 ◽  
Author(s):  
A. R. P. Paterson ◽  
J. M. Oliver
Keyword(s):  
Potent Inhibitor ◽  
Facilitated Transport ◽  
Human Erythrocytes ◽  
Nucleoside Transport ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Uridine Uptake ◽  
Related Compounds

p-Nitrobenzylthioguanosine (NBTGR) was found to be a potent inhibitor of nucleoside transport by human erythrocytes; initial rates of uridine uptake were reduced to zero upon exposure of cells to 10−6 M NBTGR. The inhibitor was firmly bound because repeated washing did not restore uridine transport capability to NBTGR-treated cells. NBTGR inhibited the influx of uridine, inosine, and cytidine, without inhibiting the uptake of the corresponding bases, or that of D-glucose or L-leucine. Uridine antagonized the NBTGR inhibition of uridine transport in a concentration-dependent manner. NBTGR and related compounds appear to interact with the mechanism for the facilitated transport of nucleosides.

Characterization of nucleoside transport activity in rabbit cortical synaptosomes

1995 ◽  
Vol 73 (12) ◽  
pp. 1733-1741 ◽  
Author(s):  
Kenneth W. James ◽  
James R. Hammond
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Mammalian Species ◽  
Nucleoside Transport ◽  
Rabbit Brain ◽  
Facilitated Diffusion ◽  
Nucleoside Transporter ◽  
Uridine Uptake ◽  
Diffusion Systems ◽  
Total Transport

Rabbit central nervous system (CNS) preparations have been used to study the central effects of adenosine, but little is known about the specific uptake mechanisms in rabbit brain involved in the regulation of extracellular adenosine concentrations. The present study assessed the kinetic and pharmacological characteristics of the uptake of [3H]uridine (a poorly metabolized substrate for adenosine transporters) by rabbit cortical synaptosomes, to define the transporter subtypes involved and to evaluate species variability in transporter characteristics. [3H]Uridine transport into rabbit cortical synaptosomes was mediated by two saturable, facilitated diffusion systems with characteristics compatible with the es and ei transporter subtypes identified in other mammalian species. About 65% of the total transport was mediated by the es system, and Km estimates of 320 and 94 μM were determined for [3H]uridine uptake by the es and ei transporter, respectively. These results differ significantly from the subtype ratio and kinetic characteristics reported for rat and guinea pig cortical synaptosomes, where most of the transport was mediated by an ei subtype. Dipyridamole, dilazep, nitrobenzylthioinosine, R75231, solufiazine, and mioflazine were relatively more effective as inhibitors of es-mediated uptake (compared with ei), while the substrates adenosine, cytidine, and guanosine did not distinguish between the es and ei transporters in rabbit cortical synaptosomes. These results highlight the significant species–tissue variability in nucleoside transporter characteristics and subtype expression, and emphasize the need to characterize the transporters in human CNS tissue to allow the rational development of CNS-active therapeutics based on inhibition of nucleoside transport.Key words: nitrobenzylthioinosine, [3H]uridine, nucleoside transport, central nervous system, adenosine.

scholarly journals Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents

1985 ◽  
Vol 232 (3) ◽  
pp. 681-688 ◽  
Author(s):  
J A Belt ◽  
L D Noel
Keyword(s):  
Diffusion Mechanism ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Lymphoma Cells ◽  
Transport Inhibitor ◽  
Mouse Lymphoma Cells ◽  
Thiol Reagent ◽  
Walker 256 ◽  
Nucleoside Transport Inhibitor ◽  
Mouse Lymphoma

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.

scholarly journals Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds

1991 ◽  
Vol 279 (2) ◽  
pp. 495-501 ◽  
Author(s):  
F E Podestá ◽  
W C Plaxton
Keyword(s):  
Pyruvate Kinase ◽  
Castor Oil ◽  
Mixed Type ◽  
Competitive Inhibition ◽  
Ricinus Communis ◽  
Cytosolic Ph ◽  
Oil Seeds ◽  
Saturation Kinetics ◽  
Regulatory Properties

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

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

1986 ◽  
Vol 236 (3) ◽  
pp. 665-670 ◽  
Author(s):  
W P Gati ◽  
J A Belt ◽  
E S Jakobs ◽  
J D Young ◽  
S M Jarvis ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cells ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Animal Cells ◽  
Novikoff Hepatoma

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.

scholarly journals PnuT uses a facilitated diffusion mechanism for thiamine uptake

2017 ◽  
Vol 150 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Michael Jaehme ◽  
Rajkumar Singh ◽  
Alisa A. Garaeva ◽  
Ria H. Duurkens ◽  
Dirk-Jan Slotboom
Keyword(s):  
Substrate Binding ◽  
Diffusion Mechanism ◽  
Vitamin B1 ◽  
Pyridine Nucleotide ◽  
Thiamine Pyrophosphate ◽  
Facilitated Diffusion ◽  
Nicotinamide Riboside ◽  
Thiamine Transport ◽  
Shewanella Woodyi ◽  
Thiamine Analogues

Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

scholarly journals Real sequence effects on the search dynamics of transcription factors on DNA

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Maximilian Bauer ◽  
Emil S. Rasmussen ◽  
Michael A. Lomholt ◽  
Ralf Metzler
Keyword(s):  
Transcription Factors ◽  
Target Detection ◽  
Diffusion Mechanism ◽  
Coli Strain ◽  
Dna Looping ◽  
Real Sequence ◽  
Facilitated Diffusion ◽  
E Coli ◽  
Sliding Motion ◽  
Sequence Effects

Abstract Recent experiments show that transcription factors (TFs) indeed use the facilitated diffusion mechanism to locate their target sequences on DNA in living bacteria cells: TFs alternate between sliding motion along DNA and relocation events through the cytoplasm. From simulations and theoretical analysis we study the TF-sliding motion for a large section of the DNA-sequence of a common E. coli strain, based on the two-state TF-model with a fast-sliding search state and a recognition state enabling target detection. For the probability to detect the target before dissociating from DNA the TF-search times self-consistently depend heavily on whether or not an auxiliary operator (an accessible sequence similar to the main operator) is present in the genome section. Importantly, within our model the extent to which the interconversion rates between search and recognition states depend on the underlying nucleotide sequence is varied. A moderate dependence maximises the capability to distinguish between the main operator and similar sequences. Moreover, these auxiliary operators serve as starting points for DNA looping with the main operator, yielding a spectrum of target detection times spanning several orders of magnitude. Auxiliary operators are shown to act as funnels facilitating target detection by TFs.

Carrier transport of glucose between blood and cerebrospinal fluid

1964 ◽  
Vol 206 (4) ◽  
pp. 836-844 ◽  
Author(s):  
Robert A. Fishman
Keyword(s):  
Cerebrospinal Fluid ◽  
Carrier Transport ◽  
Competitive Inhibition ◽  
Counter Flow ◽  
Concentration Difference ◽  
Simple Diffusion ◽  
Glucose Transfer ◽  
Saturation Kinetics ◽  
Anesthetized Dogs ◽  
Rapid Transfer

The transfer of glucose, 2-deoxy-d-glucose, and fructose between blood and cerebrospinal fluid (CSF) of anesthetized dogs was studied, following either intravenous injection or intracisternal administration in artificial CSF. Glucose entry was not directly proportional to the concentration difference between blood and CSF. The CSF glucose concentration approached a maximum, despite increasing intravenous loads, suggesting saturation kinetics. Bidirectional molecular stereospecificity was demonstrated; the three hexoses enter and exit from CSF at increasing rates, fructose < glucose < 2-deoxyglucose. The slow entry of fructose is attributed to simple diffusion. The more rapid transfer of 2-deoxyglucose than of glucose suggests greater "affinity" of the former for the carrier. Bidirectional competitive inhibition between glucose and 2-deoxyglucose was demonstrated. Bidirectional countertransport ("uphill transport by counter flow") of glucose between the two compartments was shown. Glucose transfer into CSF was not affected by intravenous 30% urea. The data support the existence of a carrier transport system for glucose in membranes separating the two compartments.

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