Hypoxanthine transport in mammalian cells: Cell type-specific differences in sensitivity to inhibition by dipyridamole and uridine

Summary We have measured by rapid kinetic techniques the zero-trans influx of hypoxanthine in various cell lines and its sensitivity to inhibition by uridine, dipyridamole, nitrobenzylthioinosine and nitrobenzylthiopurine. The results and those reported earlier divided the cells into two distinct groups. In mouse P388, L1210 and L929 cells uridine and hypoxanthine had little effect on the transport of each other, supporting the view that nucleosides and hypoxanthine are transported by different carriers. In these cells, hypoxanthine transport was also uniquely resistant to inhibition by dipyridamole (IC₅₀ (50% inhibition dose) >30M). In Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich ascites tumor cells, on the other hand, hypoxanthine and uridine inhibited the transport of each other about 50% at a concentration corresponding to the Michaelis-Menten constant of their transport, and hypoxanthine transport was strongly inhibited by dipyridamole (IC₅₀=100 to 400nM). Although these results are compatible with the view that nucleosides and hypoxanthine are transported by a common carrier in these cells, this conclusion is not supported by the finding that uridine transport is strongly inhibited in some of these cell lines, as in first group of cells, by nitrobenzylthioinsine, whereas hypoxanthine transport is highly resistant in all cell lines tested. In contrast, the transport of both substrates is highly resistant to inhibition by nitrobenzylthiopurine. The Michaelis-Menten constants for uridine transport are about the same in all cell lines. The Michaelis-Menten constants for hypoxanthine transport are similar to those for uridine transport in some cell lines, but are much higher in others. This difference is unrelated to the sensitivity of uridine and hypoxanthine transport to inhibition by each other or dipyridamole.     

Isolation by tritium suicide of uridine-cytidine kinase-defective mutants in Chinese hamster V79 cells

Tritium suicide was shown to be a highly effective method for isolating mutants defective in uridine-cytidine kinase in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of three kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [³H]uridine for 10 min, followed by storage of ³H-labelled cells at −70 °C for 4–7 days. Nine clones that survived the third kill cycle were tested for incorporation of [³H]uridine and for uridine kinase activity in extracts. Eight of these clones were defective in whole-cell uridine incorporation and in uridine kinase activity. A kinetic study was made on the uridine-cytidine kinase activity of three of the mutants. The apparent V_max of the mutants was reduced approx. 10-fold when either uridine or cytidine was used as substrate. In contrast, the apparent K_m of uridine was reduced approx. 12-fold in the mutants with only a 2-fold (probably insignificant) reduction in K_m's for cytidine or for ATP.     

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [³H]hypoxanthine for 5 or 10 min, followed by storage of ³H-labelled cells at ?70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [³H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K_m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.     

Effect of CO2 on labelling of nerve and liver cells by nucleic acid precursors

In vivo experiments in mice demonstrated that 5% CO₂ content in the air inhaled did not change the labelling in autoradiograms from animals injected with [³H]uridine, [³H]orotic acid, [³H]hypoxanthine, [³H]lysine or [³H]cytidine. At 20% CO₂ content there was a significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine, but not following [³H]lysine; there was no labelling of nerve cells with [³H]orotic acid or [³H]hypoxanthine, but a control group was not included. The labelling of choroid plexus and hepatocytes was independent of the CO₂ concentration. A comparison of in vivo and in vitro experiments at 20% CO₂ content showed a similar significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine. It is concluded that a metabolic change is the most appropriate explanation of the CO₂ effect.     

Independent blood-brain barrier transport systems for nucleic acid precursors

The blood-brain barrier permeability to certain ¹⁴C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15 s later. The amount of ¹⁴C-labelled base or nucleoside remaining in brain was expressed in relation to ³H₂O (a highly diffusible internal standard) and ^113mIn-labelled EDTA (an essentially non-diffusible internal standard).Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine.Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (K_m = 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (K_m = 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [¹⁴C]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

The existence of a group translocation transport mechanism in animal cells: Uptake of the ribose moiety of inosine

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV -40-transformed Balb/c 3T3 cells accumulate intravesicular ribose 1-PO₄ at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO₄. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO₄ neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nuceloside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO₄ site is only on the inner surface.     

Nucleoside transport in cultured mammalian cells multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 μM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine (NBTI) in a biphasic manner; 60–70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID₅₀ = 3?10 nM) and is designated NBTI-sensitive transport. The residual transport activity, designated NBTI-resistant transport, was inhibited by NBTI only at concentrations above 1 μM (ID₅₀ = 10?50 μM). S49 cells exhibited only NBTI-sensitive uridine transport, whereas Novikoff cells exhibited only NBTI-resistant uridine transport. In all instances NBTI-sensitive transport correlated with the presence of between 7·10⁴ and 7·10⁵ high-affinity NBTI binding sites/cell (K_d = 0.3?1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI-resistant and NBTI-sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI-sensitive and an NBTI-resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI. The latter site seems to be unavailable in NBTI-resistant transporters. The proportion of NBTI-resistant and sensitive uridine transport was constant during proportion of NBTI-resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Uridine uptake and its intracellular phosphorylation during the cell cycle

The rate of 5-³H uridine uptake into Chinese hamster V79 cells and the rate of its incorporation into RNA increase tenfold during the cell cycle. Both reactions exhibit the same apparent K_m(1.7 × 10^?5 M ). Chromatography of acid-soluble material from cells incubated with 5-³H uridine (0.25 μM) at different times of the cell cycle revealed that intracellular uridine was rapidly phosphorylated at all times, even though cells in late S and G₂ take up roughly ten times as much uridine as cells in G₁. Uridine kinase activity in synchronized cells increases about two and one-half-fold during the same time period, and in exponentially growing cells is not saturated until the external uridine concentration is raised above 200 μM. It is concluded that the change in uridine kinase activity during the cell cycle is not responsible for the tenfold increase in the rate of uridine transport, and that these two processes are independently regulated.     

Adverse effects of hyperbaric oxygen on [3H]uridine incorporation and uridine kinase activity in B104 rat neuroblastoma cells

The effects of hyperbaric oxygen on uracil nucleotide metabolism in B104 rat neuroblastoma cells were investigated. Cells exposed to 10 atm O₂ for 4 h incorporated markedly less [³H]uridine into the acid-soluble fraction and RNA compared to cells kept in ambient air. The acid-soluble fraction of the oxygen-treated cells contained less total [³H]uridine phosphates ([³H]UMP + [³H]UDP + [³H]UTP) than air-treated cells. Uridine kinase activity, assayed in cytosolic extracts from cells exposed to 10 atm O₂ for 4 h, was decreased by 46% compared to the air controls. The reduced enzyme activity which appears to account for the depressed [³H]uridine incorporation, may contribute to the lethal effects of oxygen in these cells.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

Cyclic AMP,membrane transport and cell division. I. Effects of various chemicals on cyclic amp levels and rate of transport of nucleosides,hypoxanthine and deoxyglucose in several lines of cultured cells

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level induced in several lines of cultured cells (normal 3T3 and SV₄₀ and polyomavirus-transformed 3T3 cells; 3T6, C6 glioma, mouse L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostagladine E, or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.     

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

Summary The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10^-6 to 19×10^-6 and the mean value was 4.1×10^-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10^-6 to 1.8×10^-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.Paper No. 1558 from the Laboratory of Genetics.Supported by N.I.H. Grants GM-06983 and GM-15422 and by a grant from the Food Research Institute of The University of Wisconsin, Madison, Wisconsin.Supported by Grant He 753-1 from Die Deutsche Forschungsgemeinschaft.     

Evaluation of Uridine Metabolism in Human and Animal Spermatozoa

The objective of this study was to elucidate the role of uridine for spermatozoa, since this pyrimidine nucleoside was found in millimolar concentration in human seminal plasma. Here, the degradative activity of uridine-phosphorylase [EC 2.4.2.3] and the salvage activity of uridine kinase [EC 2.7.1.48] were detected in human spermatozoa. HPLC analysis depicted the uptake of exogeneous ¹⁴C-labelled adenine, but not of uridine and of hypoxanthine, into nucleotide pools of boar spermatozoa. On addition of uridine, the computer-assisted semen analysis (CASA) of human cells revealed a reduction of the percentage of motile spermatozoa in contrast to an elevation of some velocity parameters. It is concluded that exogeneous uridine could function as suppressor for early capacitation and as a substrate for phosphorolysis, if ribose is needed, rather than to satisfy a demand for intracellular pyrimidine nucleotides.     

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH₁C₁) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10^?4 M cycloheximide of MH₁C₁ cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10^?4 M cycloheximide it was reduced to 83% and 67% of controls in MH₁C₁ cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10^?7 M, with 50% inhibition in MH₁C₁ cells at 4 · 10^?7 M after 12 h preincubation and at 10^-6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10^?4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.     

Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.