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At least 163 records · Page 9Linked to original sources
PubMed1989

Immunochemical and catalytic characteristics of adenosine kinase from Leishmania donovani.

Polyclonal antibodies to homogeneous preparation of adenosine kinase from Leishmania donovani were raised in rabbit. The antiserum was inhibitory and precipitated enzyme activity from both homogeneous and partially purified adenosine kinase from the parasite. However, the antiserum did not immunoprecipitate adenosine kinase of other higher eukaryotic sources tested so far. Immunoblot analysis of extracts from L. donovani and other sources revealed specific reaction of the antiserum with only the parasite enzyme. Under similar conditions, the enzyme monophosphorylated adenosine and 7-amino-3[beta-D-ribofuranosyl]-1H-pyrazolo[4,3-d]pyrimidine (formycin A) with almost equal efficiency, exhibiting Km values of 16 and 24 microM, respectively. The turnover number (Kcat) of the enzyme with both adenosine and formycin A was 24 s-1, whereas Kcat/Km yielded values of 1.5 and 1.0 microM-1 s-1, respectively. Substrate competition experiments indicated strong inhibition of [3H]formycin A phosphorylation by adenosine. In contrast, [3H]adenosine phosphorylation was insensitive to formycin A except at very high concentrations. The inhibitions of [3H]formycin A and [3H]adenosine phosphorylation by adenosine and formycin A were noncompetitive with respect to each other. Of the two nucleosides, adenosine was found to be effective in eluting the enzyme from the 5'-AMP Sepharose 4B column. Phosphorylation of [3H]formycin A was strongly inhibited by N-ethylmaleimide at concentrations which exerted minimal effect on [3H]adenosine phosphorylation. Adenosine exclusively, but not formycin A, protected the enzyme from N-ethylmaleimide-mediated inactivation. Taken together the results suggest that (a) adenosine kinase from L. donovani is immunologically distinct and (b) the enzyme possibly has two discrete catalytically active nucleoside interacting sites.

Adenosine↗
PubMed1991

Sodium-dependent nucleoside transport in rabbit intestinal epithelium.

Cellular uptake of formycin B, a poorly metabolized analog of inosine, by the isolated epithelium of rabbit jejunum is three times higher in the presence of Na+ than without this cation. The Na(+)-dependent nucleoside transport system is located in the apical membrane of the enterocytes and is capable of uphill transport, as shown for formycin B and adenosine with brush border membrane vesicles. According to present and earlier evidence, nucleoside transport across the basolateral membrane appears to have the properties of facilitated diffusion. Na(+)-dependent formycin B transport activity in intestinal epithelium decreases from jejunum to ileum and is absent in descending colon. As with Na(+)-coupled cotransport systems for other organic solutes, apical entry of formycin B is driven by the electrochemical Na+ gradient into the cell. In contrast to the facilitated diffusion system for nucleosides, Na(+)-dependent formycin B transport is not inhibited by nitrobenzylthioinosine, but both carrier systems are sensitive to inhibitors of D-glucose transport. Natural purine nucleosides and uridine are strong inhibitors of Na(+)-dependent formycin B transport. Transepithelial flux measurements substantiated that the Na(+)-dependent transport mechanism for formycin B functions as an absorptive system.

Animals↗
PubMed1985

Action of tubercidin and other adenosine analogs on Schistosoma mansoni schistosomules.

The incorporation of the radiolabeled adenosine analogs tubercidin, formycin A, 9-deaza-adenosine, and adenine arabinoside into nucleotides of Schistosoma mansoni schistosomules was studied in vitro. Of the four analogs, only tubercidin and formycin A were incorporated into the nucleotide pool, at rates respectively one-tenth and one-fiftieth the rate of adenosine incorporation. Tubercidin inhibited schistosomule motility in vitro with an approximate IC50 value of 1 microM, whereas formycin A exerted no visible effect even when more of it than of tubercidin was incorporated into the nucleotides and nucleic acids. Formycin A thus acts like a nontoxic adenosine analog. 7-Deaza-adenine, the purine base of tubercidin, was not incorporated into nucleotides. 7-Deaza-adenine, 9-deaza-adenosine, and adenine arabinoside all had no effect on schistosomule motility at concentrations up to 100 microM. Formycin A blocked the incorporation of tubercidin and of adenosine with equal effectiveness, as did p-nitrobenzyl-6-mercaptopurine ribonucleoside, a specific inhibitor of nucleoside transport in many mammalian cells. Thus, formycin A, tubercidin, and adenosine appear to have a common mechanism of cellular uptake. The significant levels of adenosine phosphorylase and adenine phosphoribosyl transferase activity found in schistosomule extracts suggests that most of the transported adenosine is converted to adenine before conversion to AMP. The levels of adenosine kinase and tubercidin kinase, while low, can more than account for the rate of tubercidin incorporated into intact schistosomules. The kinase(s) may also represent a minor pathway for direct adenosine incorporation. It may have a rather unusual substrate specificity because it is able to recognize adenosine, tubercidin, and formycin A as substrates, but not 9-deaza-adenosine or adenine arabinoside.

Adenosine↗
PubMed1990

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells.

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.

Animals↗
PubMed1990

Na(+)-dependent, active nucleoside transport in mouse spleen lymphocytes, leukemia cells, fibroblasts and macrophages, but not in equivalent human or pig cells; dipyridamole enhances nucleoside salvage by cells with both active and facilitated transport.

Formycin B influx studies have shown that P388 and L1210 mouse leukemia cells, mouse L929 cells, mouse RAW 309 Cr.1 cells, LK35.2 mouse B-cell hybridoma cells and cultured mouse peritoneal macrophages express both Na(+)-dependent, active and nonconcentrative, facilitated nucleoside transport systems. In the mouse cell lines, active transport represented only a minor nucleoside transport component and was detected only by measuring formycin B uptake in the presence of dipyridamole or nitrobenzylthioinosine, strong inhibitors of facilitated, but not of active, nucleoside transport. Inhibition of facilitated transport resulted in the concentrative accumulation of formycin B in cells expressing active nucleoside transport. Concentrative formycin B accumulation was abolished by treatment of the cells with gramicidin or absence of Na+ in the extracellular medium and strongly inhibited by ATP depletion or ouabain treatment. Mouse macrophages accumulated formycin B to 70-times the extracellular concentration in the absence of dipyridamole during 90 min of incubation at 37 degrees C. Thus active transport represents a major nucleoside transport system of these cells, similarly as previously reported for mouse spleen lymphocytes. In contrast to the various types of mouse cells, active formycin B transport was not detected in human HeLa cells, human H9, Jurkat and CEM T lymphoidal cells and pig spleen lymphocytes. These cells expressed only facilitated nucleoside transport with kinetic properties similar to those of the facilitated transporters of other mammalian cells.

Animals↗
PubMed1992

Nucleoside transport in brush border membrane vesicles from human kidney.

The goal of this study was to elucidate the mechanisms of nucleoside transport in the brush border membrane of the human kidney. [3H]Uridine was transported into brush border membrane vesicles (BBMV) from human kidney via Na(+)-independent and Na(+)-dependent processes. The Na(+)-dependent transport was saturable (Km = 4.76 +/- 0.39 microM; Vmax = 6.42 +/- 0.17 pmol/mg proteins per s) and was trans-stimulated by unlabeled uridine. Structural analogs of uridine (100 microM), 2'-deoxyuridine (2-dU) and dideoxyuridine (ddU), significantly inhibited Na(+)-uridine uptake into BBMV. Previous studies have suggested that Na(+)-nucleoside co-transport occurs via two major systems (Vijayalakshmi et al. (1988) J. Biol. Chem. 263, 19419-19423). One system (cit) is generally pyrimidine-selective; thymidine serves as a model substrate. The other system (cif) is generally purine-selective; formycin B serves as a model substrate. Uridine and adenosine are substrates of both systems. Thymidine and cytidine (100 microM), but not formycin B (100 microM) inhibited Na(+)-uridine uptake. In addition, [3H]thymidine exhibited an Na(+)-driven overshoot phenomenon whereas [3H]formycin B did not. Na(+)-thymidine uptake was inhibited by (100 microM) adenosine, uridine, guanosine, but not by formycin B and inosine. Further studies demonstrated that guanosine trans-stimulated thymidine uptake suggesting that guanosine and thymidine share a common transporter in the human renal BBMV. A different pattern was identified in BBMV from the rabbit kidney where both [3H]thymidine and [3H]formycin B as well as [3H]uridine exhibited a transient Na(+)-driven overshoot phenomenon. Collectively, these data suggest that in rabbit renal BBMV both cif and cit systems are present whereas in human renal BBMV, there appears to be a single concentrative Na(+)-nucleoside cotransport system that interacts with uridine, cytidine, thymidine, adenosine and guanosine but not with formycin B and inosine. The system is similar to the previously described cit system except that guanosine is also a substrate.

Animals↗
PubMed1975

Guanosine metabolism and regulation of fruiting body construction in dictyostelium discoideum.

A cell aggregate of Dictyostelium discoideum either constructs a fruiting body directly or transforms into a migrating slug and fruits later on in some other locale. In the presence of formycin B, an inosine analog, and in an environment that otherwise favors fruiting, aggregates having reached a relatively late (17 hr) stage of fruit construction abandon that program and transform into migrating slugs. They then revert to the fruiting mode and construct normal fruiting bodies without further interference [Brackenbury et al. (1974) J. Mol. Biol. 90, 529-539]. The data presented here suggest that formycin B exerts its morphogenetic effect by interfering competitively with the metabolism of guanosine. Thus: see article. The recovery from formycin B is thought to result from the ensuing accumulation of guanosine and reversal of the inhibition. In support of this are the following: (1) Formycin B does cause, in vivo, an accumulation of guanosine. Exogenoug guanosine reverses the effect of formycin B, depending on their relative concentrations. (2) Guanosine is phosphorylitically cleaved to guanine and ribose-1-P by purine ribonucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyl transferase, EC 2.4.2.1), present in D. discoideum extracts, and formycin B is a competitive inhibitor or this reaction with a very high affinity for the enzyme. (3) Four other analogs, also competitive inhibitors of this enzyme, produce precisely the same morphogenetic deviation. The concentrations required are consistent with the relative K1 values.

Antibiotics, Antineoplastic↗
PubMed1984

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity.

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.

Adenosine↗
PubMed1986

Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation.

Streptomyces sp. MA406-A-1 produced formycin (a nucleoside antibiotic) in parallel with cell growth in a synthetic medium. When the synthetic medium was supplemented with 1% (w/v) Casamino acids, however, formycin was produced only after the end of exponential growth. The intracellular ppGpp pool increased gradually towards the end of exponential growth and was maximal at the beginning of formycin production. After shift down from Casamino acids medium to synthetic medium, the ppGpp pool increased immediately, while the GTP pool decreased; under such conditions, the ability to produce formycin increased eightfold. Relaxed (rel) mutants, the first isolated for a Streptomyces species, were found at high incidence (10%) among spontaneous thiopeptin-resistant isolates and had severely reduced abilities to accumulate ppGpp. These rel mutants also failed to produce formycin under the usual culture conditions and exhibited numerous pleiotropic effects such as an inability to produce melanin and an extended delay of aerial mycelium formation. Thus Streptomyces sp. exhibited a typical stringent response, and the response initiated (or was needed for) the induction of secondary metabolism. The response may have also participated in the initiation of aerial mycelium formation by decreasing the intracellular GTP pool.

Amino Acids↗
PubMed1984

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.

Animals↗
PubMed1988

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities.

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.

Animals↗