JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 839-847 Copyright 0 1977 American Society for Microbiology
Vol. 131, No. 3 Printed in U.S.A.
Allantoin Transport in Saccharomyces cerevisiae ROBERTA SUMRADA AND TERRANCE G. COOPER* Department of Life Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received for publication 21 March 1977
Allantoin uptake in both growing and resting cultures of Saccharomyces cerevisiae occurs by a low-Km (ca. 15 uM) transport system that uses energy that is likely generated in the cytoplasm. This conclusion was based on the observation that transport did not occur in the absence of glucose or the presence of dinitrophenol, carbonyl cyanide-m-chloro-phenyl hydrazone, fluoride, or arsenate ions. Nornal uptake was observed, however, in the presence of cyanide. The rate of accumulation was maximal at pH 5.2. In contrast to the urea transport system, allantoin uptake appeared to be unidirectional. Preloaded, radioactive allantoin was not lost from cells suspended in allantoin-free buffer and did not exchange with exogenously added, nonradioactvie allantoin. Treatment of preloaded cells with nystatin, however, released the accumulated radioactivity. Allantoin accumulated within cells was isolated and shown to be chemically unaltered. Saccharomyces cerevisiae can utilize allantoin as sole nitrogen source by degrading it to ammonia, C02, and glyoxylate (4, 10, 17). Although there is a reasonable understanding of the biochemistry, genetics, and physiology of allantoin degradation in yeast, little information is available concerning how allantoin pathway intermediates enter the cell. The only uptake system studied so far is that responsible for urea accumulation. Here uptake can occur by either of two routes (5, 13). The first is a lowKi, inducible and repressible, active transport system that appears to be driven by cytoplasmically generated adenosine 5'-triphosphate, perhaps in association with a proton-motive force. Urea accumulated by this sytem equilibrates rapidly with extracellular urea and is quickly lost from the cell upon addition of dinitrophenol. The second mode of urea uptake occurs via an apparently constitutive, energy-independent facilitated diffusion system. Our deficiency of infornation concerning other uptake systems associated with the degradation of allantoin and related metabolites prompted us to investigate the means by which allantoin enters a yeast cell. (A preliminary report of this work has already appeared [Genetics Suppl., p. 74s, 1976].) MATERIALS AND METHODS Strains. All strains used in this work were prototrophic diploid organisms. Strain M25 is our standard wild type and was prepared as described earlier (16). Strain M85 was used for all experiments involving allantoin transport. This organism is homozygous for a mutation of the dall locus. It has only
5% of wild-type allantoinase activity, but is normal with respect to other allantoin system functions. Its genetic and biochemical characteristics have been described (10). Strain M62 lacks urea carboxylase activity (durl) (17). Culture conditions. The medium used throughout these experiments was that of Wickerham (19). Glucose (0.6%) and ammonia (0.1%) were added as sole sources of carbon and nitrogen, respectively. Cell density measurements were made with a KlettSummerson colorimeter (500- to 570-nm bandpass filter). One hundred Klett units is equivalent to about 3 x 107 cells/ml of culture. Resting-cell cultures were prepared as follows. Cells were grown to a density of about 40 Klett units, harvested by filtration, washed with several volumes of nitrogen-free medium, and suspended in one-half the original volume of prewarmed, preaerated buffer. Buffer consisted of 0.1 M citrate (pH 5.1) containing 0.6% glucose. (A pH 3.3 buffer was used in place of the pH 5.1 solution for those experiments depicted in Fig. 4B and D.) After incubation at 30°C for 18 to 24 h, the culture was ready for use. The cell density at this time was approximately 150 to 210 Klett units. These cells were used directly for the transport assays. Transfer of cells from one medium to another. In a number of the experiments, we had to transfer cell samples from one medium to another. This was done by filtering the culture through membrane filters and suspending the harvested cells in fresh medium. All filtrations were performed with nitrocellulose filters (0.45-j,m pore diameter; Millipore Corp.) and were completed in less than 15 to 20 s. The extent of cell loss during this procedure was determined by using radioactively labeled cells and was found to be negligible (2). Allantoin uptake assay. At zero time, an 8.5-ml portion of the culture to be assayed was transferred to a prewarmed flask containing 0.3 to 0.9 mM 839
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['4C]allantoin (specific activity, 0.2 ,uCi/iLmol). This concentration of allantoin was 25- to 75-fold greater than the apparent Michaelis constant of the transport system. Incubation was carried out at 30°C in a shaking water bath under conditions identical to those used for growth. At the times indicated, 1.0-ml samples were removed and transferred to nitrocellulose membrane filters. These filters were then washed five times with 4 ml of cold minimal medium containing 0.1% allantoin. The temperature of the wash solution did not appear to be significant, since the same values of accumulation were observed when cells were washed with medium at either 27 or 4°C. Washed filters were placed in 5 ml of aqueous scintillation fluid (Aquasol, New England Nuclear Corp.), and their radioactivity content was determined 16 to 24 h later. The incubation time in Aquasol was needed to allow the filters to become transparent. Failure to do this resulted in unevenly quenched samples and loss of assay precision. All data are expressed as amounts of radioactive allantoin accumulated per milliliter of culture unless otherwise indicated. It should be noted that restingcell cultures accumulated more allantoin than growing cells. This difference results in part from the greater cell densities of resting-cell cultures. Synthesis of radioactive allantoin. [14C]allantoin was synthesized by condensing [14C]urea and diethoxyacetic acid ethyl ester under acidic conditions with standard procedures (C. N. Zellner and J. R. Stevens, pat. 2158098, 1939). The product was recrystallized once from hot water and dried in vacuo at 4°C. The final product was obtained at an overall yield of 31%. The specific activity of allantoin was determined by carefully weighing a sample, dissolving it in a precisely known volume of water, and measuring the radioactivity content of the solution. Paper chromatography. Compounds to be analyzed were spotted on Whatman no. 1 medium-flowgrade filter paper. The chromatogram was developed for approximately 16 h in a descending orientation with butanol-acetic acid-water (100:22:50) as the solvent system. After development, the paper was air dried in a fume hood and then either cut into 0.5cm strips for radioactivity determination or sprayed with Ehrlich reagent (12) for visual observation of ureido-containing compounds. Ion-exchange chromatography. Ion-exchange resins were prepared as described by Cooper and Beevers (3) and equilibrated with the indicated counterions. Conditions of anion-exchange chromatography are summarid in the appropriate legends. Cation-exchange chromatography was performed as described by Watts et al. (15). Dowex-50 resin was equilibrated with 0.1 N HCI. Samples were dissolved in 0.1 N HCI and allowed to pass through the resin. The resin was then washed with several volumes of 0.1 N HCI. Urea was removed from the resin with a 4 M solution of sodium chloride dissolved in 0.1 N HCI.
RESULTS Purity and authenticity of radioactive allantoin. Our characterization of the allantoin transport system in S. cerevisiae was based
J. BACTERIOL.
upon the observed intracellular accumulation of [14C]allantoin. It was therefore important to
demonstrate the authenticity and purity of our allantoin preparations. This was done in several ways. First, the preparation was shown to be free of urea by the following experiment. A sample of our radioactive material was dissolved in water and passed over a Dowex-50-X-8 ion-exchange resin (free-proton form 0.1 N HCl). All of the radioactivity was recovered in the unadsorbed eluate, the expected behavior for allantoin. Urea, a likely contaminant, would have remained bound to the resin (15); no bound material was observed after a highsalt wash, indicating that the preparation was free of urea. To show that the prepared material was free of allantoic acid, this experiment was repeated with Dowex-1-X-8-acetate ion-exchange resin. Again, all of the radioactive material was found in the unadsorbed eluate, indicating that the preparation was free ofallantoic acid and negatively charged side products usually produced during the condensation reaction. Next, the radioactive preparation was incubated with 0.5 N KOH, a treatment known to convert allantoin to potassium allantoate (21). After this treatment, the radioactive product was quantitatively eluted from Dowex-l-acetate resin coincident with authentic nonradioactive potassium allantoate. The material we prepared also co-migrated as a single species with authentic allantoin on a paper chromatogram (Fig. 1, inset). Potassium allantoate and urea standards were included in this analysis, and neither of these compounds was present (Fig. 1). Finally, samples of authentic and prepared allantoin were subjected to mass spectrometric analysis. In this analysis, both samples exhibited identical molecular ions and fragmentation patterns. Collectively, these data suggest that the radioactive material we prepared was allantoin, free of other allantoin pathway intermediates. Another test of identity was made with mutant strains of Saccharomyces previously shown to lack various enzyme activities of the allantoin degradative pathway. Our [14C]allantoin preparation was efficiently degraded to "4CO2 by wild-type organisms (Fig. 1). However, little radioactive CO2 was evolved when the synthesized material was added to cultures possessing either defective allantoinase (dall) or urea carboxylase (durl). Allantoin accumulation during the transition of cells from growing to resting states. Solute uptake may be rigorously studied only when other cellular processes, such as cell division or gross changes in metabolism, are held constant. However, when studying the synthe-
ALLANTOIN TRANSPORT
VOL. 131, 1977
MINUTES
FIG. 1. Degradation of synthetic radioactive allantoin by wild-type and mutant strains ofS. cerevisiae. All three strains were grown to a cell density of about 25 Klett units in glucose-ammonia medium containing 0.5 mM oxaluric acid. A portion of each culture was then added to three samples ofour allantoin preparation in closed vessels. The final concentration of allantoin was 0.47 mM. At the times indicated, metabolism was terminated by addition of 02 ml of perchloric acid to the vessels. "4CO2 liberated from the cells and medium was absorbed with hyamine hydroxide for 45 min, followed by a determination ofthe amounts ofabsorbed material (16). Five percent of the allantoin initially provided was degraded by the wild-type strain during the course of this experiment. Inset: Recrystallized allantoin was dissolved in water and applied to a piece of Whatman filter paper along with authentic nonradioactive allantoin, allantoic acid, and urea. The chromatogram was then developed as described in Materials and Methods. Arrows indicate the positions of urea and allantoic acid. Radioactive material and nonradioactive allantoin (Ehrlich reagent-positive material) migrated at identical rates.
841
metabolic realignment of the cell. An early-logphase culture of strain M85 was harvested by filtration and suspended in the same volume of buffer containing 0.6% glucose as an energy source. At various times thereafter, the initial rate of allantoin accumulation (nanomoles accumulated per 10 min) was measured. The rate of uptake initially increased, oscillated, and finally damped to a constant value after about 16 to 20 h (Fig. 2). The source of observed variations in the rate of accumulation is not known. Difficulties that may have occurred due to fluctuations in the rate of allantoin accumulation were avoided by allowing cells to incu-
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sis and assembly of transport system components, it may also be advantageous to use logarithmically growing cultures. Therefore, we characterized allantoin accumulation by cells in both the growing and resting states. In resting cultures, cell division was arrested at the G-1 stage due to nitrogen starvation (Sumrada and Cooper, manuscript in preparation). We have recently shown (Cooper and Sumrada, manuscript in preparation) that a variety of complex adjustments are made by a cell suddenly faced with nitrogen deficiency. For example, arginine present in the vacuole (20) becomes metabolically available and induces formation of arginase. Action of arginase on the released arginine results in production of urea and allophanate, which in turn induces formation of the allantoin degradative enzymes (18). In view of these past observations, an experiment was performed to ascertain the rate of allantoin uptake at various times during this
FIG. 2. Rate of [14C]allantoin accumulation by cells that are proceeding from a growing to a resting state. Cultures of strain M85 were grown to a cell density of 45 Klett units in the presence (B) and absence (A) of 0.5 mM oxaluric acid. We showed later that the presence or absence of this compound had no effect upon the rate of allantoin accumulation. The significant difference between these two experiments was the length of time that accumulation was monitored. In (A), accumulation was measured at high resolution over a short period of time, whereas in (B), resolution was sacrificed to follow accumulation for a much longer time. At zero time, the cultures were harvested by filtration, washed, and resuspended in their initial volume of citrate buffer (see Materials and Methods). At the times indicated, a 1ml sample of each culture was transferred to a second small flask containing ['4C]allantoin at a final concentration of 0.9 mM. After 10 min of incubation at 30°C, 0.5 ml of the assay mixture was transferred to a membrane filter (Millipore Corp.) to separate the cells from the radioactive medium. After this point, all samples were processed as described in the text.
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bate overnight (18 to 24 h) in nitrogen-free medium before being used as a resting culture. The rate of allantoin accumulation rened constant for at least 40 h under these conditions, providing ample time to conduct our experiments (Fig. 2B). Accumulation of allantoin against a concentration gradient. Several criteria must be fulfilled to demonstrate that allantoin accumulation results fom active transport. At steady state, the intracellular allantoin concentration must be greater than that in the surrounding medium. In addition, the accumulated material must be shown to be unmodified. To ascertain whether these criteria were met, 30 IAM [14CIallantoin was added to a resting culture of Saccharomyces. Thereafter, samples were removed, and the amounts of radioactive allantoin in the cells and medium were determined. Intracellular allantoin continued to increase until virtually all of it was removed from the medium (Fig. 3). To calculate the intracellular concentration of allantoin, a standard curve was prepared that relates cell density (Klett units) to dry weight (milligrams). We found that 1 ml of culture, at a cell density of 208 Klett units, contained 1.27 mg of dxy material. This value was used to ascertain the amount of dry material in our cell samples. We assumed that the volume of a yeast cell was four times its weight (11) and estimated the intra- and extracellular concentrations of allantoin to be 4.1 mM and 0.53 yM, respectively, after 140 min of incubation. This suggests that a 7,700fold concentration of allantoin occurred during this experiment. Such efficient accumulation of solute raised the possibility that some modification of the allantoin occurred within the cell and that the modified material could not leave the cell by using the same transport system. To evaluate this possibility, soluble components were extracted with chloroform-methanol from cells that had been allowed to accumulate [14C]allantoin for a long period of time. This material was not able to bind to Dowex-1-acetate ion-exchange resin. Treatment with base, however, resulted in quantitative production of a compound that could be eluted from the resin coincident with authentic carrier allantoic acid. The elution patterns observed in this experiment were very similar to those shown in Fig. 1. As shown in the inset of Fig. 3, all of the isolated material (open circles) co-migrated with authentic allantoin (closed cicls) during paper chromatography. Non-radioactive carrier allantoin was added to a sample of the cell extract prepared above. The carrier allantoin was
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Vol. 131, No. 3 Printed in U.S.A.
Allantoin Transport in Saccharomyces cerevisiae ROBERTA SUMRADA AND TERRANCE G. COOPER* Department of Life Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received for publication 21 March 1977
Allantoin uptake in both growing and resting cultures of Saccharomyces cerevisiae occurs by a low-Km (ca. 15 uM) transport system that uses energy that is likely generated in the cytoplasm. This conclusion was based on the observation that transport did not occur in the absence of glucose or the presence of dinitrophenol, carbonyl cyanide-m-chloro-phenyl hydrazone, fluoride, or arsenate ions. Nornal uptake was observed, however, in the presence of cyanide. The rate of accumulation was maximal at pH 5.2. In contrast to the urea transport system, allantoin uptake appeared to be unidirectional. Preloaded, radioactive allantoin was not lost from cells suspended in allantoin-free buffer and did not exchange with exogenously added, nonradioactvie allantoin. Treatment of preloaded cells with nystatin, however, released the accumulated radioactivity. Allantoin accumulated within cells was isolated and shown to be chemically unaltered. Saccharomyces cerevisiae can utilize allantoin as sole nitrogen source by degrading it to ammonia, C02, and glyoxylate (4, 10, 17). Although there is a reasonable understanding of the biochemistry, genetics, and physiology of allantoin degradation in yeast, little information is available concerning how allantoin pathway intermediates enter the cell. The only uptake system studied so far is that responsible for urea accumulation. Here uptake can occur by either of two routes (5, 13). The first is a lowKi, inducible and repressible, active transport system that appears to be driven by cytoplasmically generated adenosine 5'-triphosphate, perhaps in association with a proton-motive force. Urea accumulated by this sytem equilibrates rapidly with extracellular urea and is quickly lost from the cell upon addition of dinitrophenol. The second mode of urea uptake occurs via an apparently constitutive, energy-independent facilitated diffusion system. Our deficiency of infornation concerning other uptake systems associated with the degradation of allantoin and related metabolites prompted us to investigate the means by which allantoin enters a yeast cell. (A preliminary report of this work has already appeared [Genetics Suppl., p. 74s, 1976].) MATERIALS AND METHODS Strains. All strains used in this work were prototrophic diploid organisms. Strain M25 is our standard wild type and was prepared as described earlier (16). Strain M85 was used for all experiments involving allantoin transport. This organism is homozygous for a mutation of the dall locus. It has only
5% of wild-type allantoinase activity, but is normal with respect to other allantoin system functions. Its genetic and biochemical characteristics have been described (10). Strain M62 lacks urea carboxylase activity (durl) (17). Culture conditions. The medium used throughout these experiments was that of Wickerham (19). Glucose (0.6%) and ammonia (0.1%) were added as sole sources of carbon and nitrogen, respectively. Cell density measurements were made with a KlettSummerson colorimeter (500- to 570-nm bandpass filter). One hundred Klett units is equivalent to about 3 x 107 cells/ml of culture. Resting-cell cultures were prepared as follows. Cells were grown to a density of about 40 Klett units, harvested by filtration, washed with several volumes of nitrogen-free medium, and suspended in one-half the original volume of prewarmed, preaerated buffer. Buffer consisted of 0.1 M citrate (pH 5.1) containing 0.6% glucose. (A pH 3.3 buffer was used in place of the pH 5.1 solution for those experiments depicted in Fig. 4B and D.) After incubation at 30°C for 18 to 24 h, the culture was ready for use. The cell density at this time was approximately 150 to 210 Klett units. These cells were used directly for the transport assays. Transfer of cells from one medium to another. In a number of the experiments, we had to transfer cell samples from one medium to another. This was done by filtering the culture through membrane filters and suspending the harvested cells in fresh medium. All filtrations were performed with nitrocellulose filters (0.45-j,m pore diameter; Millipore Corp.) and were completed in less than 15 to 20 s. The extent of cell loss during this procedure was determined by using radioactively labeled cells and was found to be negligible (2). Allantoin uptake assay. At zero time, an 8.5-ml portion of the culture to be assayed was transferred to a prewarmed flask containing 0.3 to 0.9 mM 839
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SUMRADA AND COOPER
['4C]allantoin (specific activity, 0.2 ,uCi/iLmol). This concentration of allantoin was 25- to 75-fold greater than the apparent Michaelis constant of the transport system. Incubation was carried out at 30°C in a shaking water bath under conditions identical to those used for growth. At the times indicated, 1.0-ml samples were removed and transferred to nitrocellulose membrane filters. These filters were then washed five times with 4 ml of cold minimal medium containing 0.1% allantoin. The temperature of the wash solution did not appear to be significant, since the same values of accumulation were observed when cells were washed with medium at either 27 or 4°C. Washed filters were placed in 5 ml of aqueous scintillation fluid (Aquasol, New England Nuclear Corp.), and their radioactivity content was determined 16 to 24 h later. The incubation time in Aquasol was needed to allow the filters to become transparent. Failure to do this resulted in unevenly quenched samples and loss of assay precision. All data are expressed as amounts of radioactive allantoin accumulated per milliliter of culture unless otherwise indicated. It should be noted that restingcell cultures accumulated more allantoin than growing cells. This difference results in part from the greater cell densities of resting-cell cultures. Synthesis of radioactive allantoin. [14C]allantoin was synthesized by condensing [14C]urea and diethoxyacetic acid ethyl ester under acidic conditions with standard procedures (C. N. Zellner and J. R. Stevens, pat. 2158098, 1939). The product was recrystallized once from hot water and dried in vacuo at 4°C. The final product was obtained at an overall yield of 31%. The specific activity of allantoin was determined by carefully weighing a sample, dissolving it in a precisely known volume of water, and measuring the radioactivity content of the solution. Paper chromatography. Compounds to be analyzed were spotted on Whatman no. 1 medium-flowgrade filter paper. The chromatogram was developed for approximately 16 h in a descending orientation with butanol-acetic acid-water (100:22:50) as the solvent system. After development, the paper was air dried in a fume hood and then either cut into 0.5cm strips for radioactivity determination or sprayed with Ehrlich reagent (12) for visual observation of ureido-containing compounds. Ion-exchange chromatography. Ion-exchange resins were prepared as described by Cooper and Beevers (3) and equilibrated with the indicated counterions. Conditions of anion-exchange chromatography are summarid in the appropriate legends. Cation-exchange chromatography was performed as described by Watts et al. (15). Dowex-50 resin was equilibrated with 0.1 N HCI. Samples were dissolved in 0.1 N HCI and allowed to pass through the resin. The resin was then washed with several volumes of 0.1 N HCI. Urea was removed from the resin with a 4 M solution of sodium chloride dissolved in 0.1 N HCI.
RESULTS Purity and authenticity of radioactive allantoin. Our characterization of the allantoin transport system in S. cerevisiae was based
J. BACTERIOL.
upon the observed intracellular accumulation of [14C]allantoin. It was therefore important to
demonstrate the authenticity and purity of our allantoin preparations. This was done in several ways. First, the preparation was shown to be free of urea by the following experiment. A sample of our radioactive material was dissolved in water and passed over a Dowex-50-X-8 ion-exchange resin (free-proton form 0.1 N HCl). All of the radioactivity was recovered in the unadsorbed eluate, the expected behavior for allantoin. Urea, a likely contaminant, would have remained bound to the resin (15); no bound material was observed after a highsalt wash, indicating that the preparation was free of urea. To show that the prepared material was free of allantoic acid, this experiment was repeated with Dowex-1-X-8-acetate ion-exchange resin. Again, all of the radioactive material was found in the unadsorbed eluate, indicating that the preparation was free ofallantoic acid and negatively charged side products usually produced during the condensation reaction. Next, the radioactive preparation was incubated with 0.5 N KOH, a treatment known to convert allantoin to potassium allantoate (21). After this treatment, the radioactive product was quantitatively eluted from Dowex-l-acetate resin coincident with authentic nonradioactive potassium allantoate. The material we prepared also co-migrated as a single species with authentic allantoin on a paper chromatogram (Fig. 1, inset). Potassium allantoate and urea standards were included in this analysis, and neither of these compounds was present (Fig. 1). Finally, samples of authentic and prepared allantoin were subjected to mass spectrometric analysis. In this analysis, both samples exhibited identical molecular ions and fragmentation patterns. Collectively, these data suggest that the radioactive material we prepared was allantoin, free of other allantoin pathway intermediates. Another test of identity was made with mutant strains of Saccharomyces previously shown to lack various enzyme activities of the allantoin degradative pathway. Our [14C]allantoin preparation was efficiently degraded to "4CO2 by wild-type organisms (Fig. 1). However, little radioactive CO2 was evolved when the synthesized material was added to cultures possessing either defective allantoinase (dall) or urea carboxylase (durl). Allantoin accumulation during the transition of cells from growing to resting states. Solute uptake may be rigorously studied only when other cellular processes, such as cell division or gross changes in metabolism, are held constant. However, when studying the synthe-
ALLANTOIN TRANSPORT
VOL. 131, 1977
MINUTES
FIG. 1. Degradation of synthetic radioactive allantoin by wild-type and mutant strains ofS. cerevisiae. All three strains were grown to a cell density of about 25 Klett units in glucose-ammonia medium containing 0.5 mM oxaluric acid. A portion of each culture was then added to three samples ofour allantoin preparation in closed vessels. The final concentration of allantoin was 0.47 mM. At the times indicated, metabolism was terminated by addition of 02 ml of perchloric acid to the vessels. "4CO2 liberated from the cells and medium was absorbed with hyamine hydroxide for 45 min, followed by a determination ofthe amounts ofabsorbed material (16). Five percent of the allantoin initially provided was degraded by the wild-type strain during the course of this experiment. Inset: Recrystallized allantoin was dissolved in water and applied to a piece of Whatman filter paper along with authentic nonradioactive allantoin, allantoic acid, and urea. The chromatogram was then developed as described in Materials and Methods. Arrows indicate the positions of urea and allantoic acid. Radioactive material and nonradioactive allantoin (Ehrlich reagent-positive material) migrated at identical rates.
841
metabolic realignment of the cell. An early-logphase culture of strain M85 was harvested by filtration and suspended in the same volume of buffer containing 0.6% glucose as an energy source. At various times thereafter, the initial rate of allantoin accumulation (nanomoles accumulated per 10 min) was measured. The rate of uptake initially increased, oscillated, and finally damped to a constant value after about 16 to 20 h (Fig. 2). The source of observed variations in the rate of accumulation is not known. Difficulties that may have occurred due to fluctuations in the rate of allantoin accumulation were avoided by allowing cells to incu-
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sis and assembly of transport system components, it may also be advantageous to use logarithmically growing cultures. Therefore, we characterized allantoin accumulation by cells in both the growing and resting states. In resting cultures, cell division was arrested at the G-1 stage due to nitrogen starvation (Sumrada and Cooper, manuscript in preparation). We have recently shown (Cooper and Sumrada, manuscript in preparation) that a variety of complex adjustments are made by a cell suddenly faced with nitrogen deficiency. For example, arginine present in the vacuole (20) becomes metabolically available and induces formation of arginase. Action of arginase on the released arginine results in production of urea and allophanate, which in turn induces formation of the allantoin degradative enzymes (18). In view of these past observations, an experiment was performed to ascertain the rate of allantoin uptake at various times during this
FIG. 2. Rate of [14C]allantoin accumulation by cells that are proceeding from a growing to a resting state. Cultures of strain M85 were grown to a cell density of 45 Klett units in the presence (B) and absence (A) of 0.5 mM oxaluric acid. We showed later that the presence or absence of this compound had no effect upon the rate of allantoin accumulation. The significant difference between these two experiments was the length of time that accumulation was monitored. In (A), accumulation was measured at high resolution over a short period of time, whereas in (B), resolution was sacrificed to follow accumulation for a much longer time. At zero time, the cultures were harvested by filtration, washed, and resuspended in their initial volume of citrate buffer (see Materials and Methods). At the times indicated, a 1ml sample of each culture was transferred to a second small flask containing ['4C]allantoin at a final concentration of 0.9 mM. After 10 min of incubation at 30°C, 0.5 ml of the assay mixture was transferred to a membrane filter (Millipore Corp.) to separate the cells from the radioactive medium. After this point, all samples were processed as described in the text.
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bate overnight (18 to 24 h) in nitrogen-free medium before being used as a resting culture. The rate of allantoin accumulation rened constant for at least 40 h under these conditions, providing ample time to conduct our experiments (Fig. 2B). Accumulation of allantoin against a concentration gradient. Several criteria must be fulfilled to demonstrate that allantoin accumulation results fom active transport. At steady state, the intracellular allantoin concentration must be greater than that in the surrounding medium. In addition, the accumulated material must be shown to be unmodified. To ascertain whether these criteria were met, 30 IAM [14CIallantoin was added to a resting culture of Saccharomyces. Thereafter, samples were removed, and the amounts of radioactive allantoin in the cells and medium were determined. Intracellular allantoin continued to increase until virtually all of it was removed from the medium (Fig. 3). To calculate the intracellular concentration of allantoin, a standard curve was prepared that relates cell density (Klett units) to dry weight (milligrams). We found that 1 ml of culture, at a cell density of 208 Klett units, contained 1.27 mg of dxy material. This value was used to ascertain the amount of dry material in our cell samples. We assumed that the volume of a yeast cell was four times its weight (11) and estimated the intra- and extracellular concentrations of allantoin to be 4.1 mM and 0.53 yM, respectively, after 140 min of incubation. This suggests that a 7,700fold concentration of allantoin occurred during this experiment. Such efficient accumulation of solute raised the possibility that some modification of the allantoin occurred within the cell and that the modified material could not leave the cell by using the same transport system. To evaluate this possibility, soluble components were extracted with chloroform-methanol from cells that had been allowed to accumulate [14C]allantoin for a long period of time. This material was not able to bind to Dowex-1-acetate ion-exchange resin. Treatment with base, however, resulted in quantitative production of a compound that could be eluted from the resin coincident with authentic carrier allantoic acid. The elution patterns observed in this experiment were very similar to those shown in Fig. 1. As shown in the inset of Fig. 3, all of the isolated material (open circles) co-migrated with authentic allantoin (closed cicls) during paper chromatography. Non-radioactive carrier allantoin was added to a sample of the cell extract prepared above. The carrier allantoin was
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