Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Cybulski, Raymond L.; Fry, David W.; Goldman, I. David

doi:10.1016/0304-4165(82)90151-9

Cited by 5 publications

(2 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the experiment shown in Fig. 4 a comparison was made between uridine uptake in cells depleted of ATP by incubation with azide (Cybulski et al, 1981) and in untreated cells. The initial rate of uridine uptake was the same in ATP-depleted and untreated cells (inset), but uptake rapidly reached a plateau in the azide-treated cells, with the intracellular concentration of 3H approximately equal to that in the medium.…”

Section: Resultsmentioning

confidence: 99%

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

Belt¹,

Noël²

1985

Biochemical Journal

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

Belt¹,

Noël²

1985

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Accumulation of its active coenzyme form is attributed to rapid phosphorylation of thiamin to TPP which is, to a large extent, retained within cells. This is a common phenomenon for many different substrates of the major facilitator superfamily, as occurs with phosphorylation of nucleosides (27,28) and polyglutamation of folates (29). Consistent with this was the observation that in energy-depleted cells, phosphorylation was impaired and net thiamin uptake was markedly decreased (Fig.…”

Section: Discussionmentioning

confidence: 99%

Impact of the Reduced Folate Carrier on the Accumulation of Active Thiamin Metabolites in Murine Leukemia Cells

Zhao¹,

Gao²,

Wang³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The thiamin transporter encoded by SLC19A2 and the reduced folate carrier (RFC1) share 40% homology at the protein level, but the thiamin transporter does not mediate transport of folates. By using murine leukemia cell lines that express no, normal, or high levels of RFC1, we demonstrate that RFC1 does not mediate thiamin influx. However, high level RFC1 expression substantially reduced accumulation of the active thiamin coenzyme, thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized intracellularly from imported thiamin, resulted from RFC1-mediated efflux of TPP. The reduced folate carrier (RFC1), 1 first cloned in 1994, mediates transport of reduced folates critical to one carbonrequiring biosynthetic reactions in mammalian cells and is a member of the major facilitator superfamily of transporters (1-3). RFC1 also delivers MTX and new generation antifolates into a variety of tumors, particularly those of hematopoietic origin (4). RFC1 exchanges folates with a broad spectrum of inorganic and organic anions, and high extracellular concentrations of a variety of organic phosphates competitively inhibit RFC1-mediated folate influx (5-7). This interaction between RFC1 and organic phosphates results in the uphill transport of folates into cells linked to the organic phosphate gradient across cell membranes (5).Structurally unrelated to the folates, thiamin plays an essential role in glycolysis and oxidative decarboxylation reactions after conversion to the coenzyme thiamin pyrophosphate by thiamin pyrophosphokinase in cells. Thiamin is also transported across cell membranes by a carrier-mediated process (8). Thiamin deficiency, reflected in a decrease in plasma thiamin concentration and TPP levels in erythrocytes, results in a variety of clinical abnormalities including cardiovascular and neurological disorders (9). Thiamin deficiency due to impaired transport results in the thiamin-responsive megaloblastic anemia syndrome, a disorder also associated with deafness and diabetes mellitus (10, 11). Positional cloning with families inheriting this autosomal recessive disease led to the recent identification of the thiamin transporter gene SLC19A2 (12-14).The thiamin transporter encoded by SLC19A2 is highly homologous to RFC1, sharing an amino acid identity of 40% and similarity of 55%, and both are predicted to have 12 transmembrane domains. Despite the similarity between these two proteins, the thiamin transporter, when expressed in HeLa cells, was not found to transport folates (15). In the current report, the impact of RFC1 function on thiamin transport and accumulation of its active coenzyme metabolites was studied in murine leukemia cells. Although RFC1 was not found to transport thiamin, it does transport phosphorylated thiamin derivatives, thereby modulating the intracellular accumulation of active thiamin metabolites. -[3Ј,5Ј,7-3 H]MTX (5.7 Ci/mmol) and [ 3 H]thiamin hydrochloride (20 Ci/mmol) were obtained from Amersham Pharmacia Biotech, and [ 3 H]TPP (generally labeled, 4.2 Ci/mmol) was custo...

show abstract

Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria

Upston

Gero

1995

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

A mechanism which mediates the transport of the nonphysiological nucleoside, L-adenosine, was demonstrated in Plasmodium falciparum infected erythrocytes and naturally released merozoites. L-Adenosine was not a substrate for influx in freed intraerythrocytic parasites or in normal human erythrocytes nor was L-adenosine transported in a variety of cell types including other parasitic protozoa such as Crithidia luciliae, Trichomonas vaginalis, Giardia intestinalis, or the mammalian cells, Buffalo Green Monkey and HeLa cells. L-Adenosine transport in P. falciparum infected cells was nonsaturable, with a rate of 0.13 +/- 0.01 pmol/microliter cell water per s per microM L-adenosine, yet the transport was inhibited by furosemide, phloridzin and piperine with IC50 values between 1-13 microM, distinguishing the transport pathway from simple diffusion. The channel-like permeation was selective as disaccharides were not permeable to parasitised cells. In addition, an unusual metabolic property of parasitic adenosine deaminase was found in that L-adenosine was metabolised to L-inosine by both P. falciparum infected erythrocytes and merozoites, an activity which was inhibited by 50 nM deoxycoformycin. No other cell type examined displayed this enzymic activity. The results further substantiate that nucleoside transport in P. falciparum infected cells was significantly altered compared to uninfected erythrocytes and that L-adenosine transport and metabolism was a biochemical property of Plasmodium infected cells and merozoites and not found in normal erythrocytes nor any of the other cell types investigated.

show abstract

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Cited by 5 publications

References 18 publications

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

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

Impact of the Reduced Folate Carrier on the Accumulation of Active Thiamin Metabolites in Murine Leukemia Cells

Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria

Contact Info

Product

Resources

About