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A study of the free amino acid pool and enzyme synthesis in yeast

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Abstract

A study has been made of the distribution and properties of the free amino acid pool in yeast. The depletion of the pool was found to depend upon the energy source used, conditions of growth, and the nature of the exogenous nitrogen source. Pool levels could be restored either by an internal replenishment mechanism or by various nitrogen sources. In the absence of internal replenishment a strong positive correlation was established between the ability of nitrogen compounds to support free glutamic add synthesis and enzyme-synthesizing capacity. Amino acid assimilation by nitrogen-starved yeast was studied and compared with that in other organisms. The significance of these results for the problem of enzyme and protein synthesis in yeast is discussed.

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... One of the earliest papers on the uptake of amino acids by a yeast was that of Halvorson and his colleagues [152] who, in 1955, published evidence that nitrogen-starved cells of Saccharomyces cerevisiae can concentrate externally-supplied arginine, glutamic acid or lysine up to 1000-fold ( Figure 6). However, most amino acid carriers were discovered by a combination of biochemical and genetic methods, many by Marcelle Grenson 13 (Figure 7) and her colleagues (Table 3). ...
... Several publications in the 1950s and 1960s reported the uptake by yeasts of various amino acids, exploiting competitive inhibitions between each of them to give kinetic evidence of wide specificity of the amino acid carrier(s) of Saccharomyces cerevisiae [151,152,357,358]. Grenson, in particular, obtained History of research on yeasts 13 699 Figure 6. ...
... Grenson, in particular, obtained History of research on yeasts 13 699 Figure 6. Uptake of amino acids by Saccharomyces cerevisiae; modified from a paper by Halvorson and his colleagues published in 1955 [152]. Nitrogen-starved cells were aerated at 30 • C and exposed to various concentrations of amino acids for 5 min. ...
... In yeast, amino acids are stored both in the cytosol and in a vacuole (Halvorson, 1955;Wiemken and Durr, 1974;Messenguy et al., 1980;Li and Kane, 2009). The regulation of these distinct, dynamic nutrient pools is complex. ...
Article
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Adaptive modulation of the global cellular growth state of unicellular organisms is crucial for their survival in fluctuating nutrient environments. Because these organisms must be able to respond reliably to ever varying and unpredictable nutritional conditions, their nutrient signaling networks must have a certain in-built robustness. In eukaryotes, such as the budding yeast Saccharomyces cerevisiae, distinct nutrient signals are relayed by specific plasma membrane receptors to signal transduction pathways that are interconnected in complex information-processing networks, which have been well characterized. However the complexity of the signaling network confounds the interpretation of the overall regulatory ‘logic’ of the control system. Here, we propose a literature-curated molecular mechanism of the integrated nutrient signaling network in budding yeast, focusing on early temporal responses to carbon and nitrogen signaling. We build a computational model of this network to reconcile literature-curated quantitative experimental data with our proposed molecular mechanism. We evaluate the robustness of our estimates of the model's kinetic parameter values. We test the model by comparing predictions made in mutant strains with qualitative experimental observations made in the same strains. Finally, we use the model to predict nutrient-responsive transcription factor activities in a number of mutant strains undergoing complex nutrient shifts.
... increase in the number of cells and continued RNA and protein synthesis until the intracellular inorganic polyphosphates are severely depleted (16). The present investigation shows that macromolecular synthesis and cell division continue for some time after removal of a required amino acid which is fairly abundant in the cell pool (6,12 which are specific for intact tRNA (7). Five of the nine strains investigated failed to accumulate methyl-deficient tRNA (Table 2); that this is due to a lack of sites on the tRNA (and not to labile methylase preparations) was shown by experiments in which enzyme and tRNA preparations from different strains were used (unpublished data). ...
Article
Haploid methionine auxotrophs of Saccharomyces cerevisiae continue to multiply for several hours after withdrawal of a required amino acid from the medium. Macro-molecular synthesis continues during this period of residual growth, although the net ribonucleic acid (RNA) and protein content is constant during the later part of this period. In this study, growth after withdrawal of methionine was in some cases accompanied by accumulation of transfer RNA (tRNA), which was shown by methylation in vitro to be deficient in methyl groups. This phenomenon was shown by only four of nine methionine auxotrophs tested, but no evidence could be found that these four strains had "relaxed" control of RNA synthesis. The nine methionine-requiring strains represent mutations in five different positions in the methionine biosynthesis pathway, and only mutants blocked at two of these five positions accumulated methyl-deficient tRNA. This accumulation therefore appears to be correlated with the position of the strain's block in the pathway of methionine biosynthesis.
... Storage molecules. In yeast, amino acids are stored both in the cytosol and in a vacuole [26,27,28,29]. ...
Preprint
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In eukaryotes, distinct nutrient signals are relayed by specific plasma membrane receptors to signal transduction pathways that are interconnected in complex information-processing networks. The function of these networks is to govern robust cellular responses to unpredictable changes in the nutritional environment of the cell. In the budding yeast, Saccharomyces cerevisiae, these nutrient signaling pathways and their interconnections have been well characterized. However the complexity of the signaling network confounds the interpretation of the overall regulatory 'logic' of the control system. Here, we propose a literature-curated molecular mechanism of the integrated nutrient signaling network in budding yeast, focusing on early temporal responses to carbon and nitrogen signaling. We build a computational model of this network to reconcile literature-curated quantitative experimental data with our proposed molecular mechanism. We evaluate the robustness of our estimates of the model's kinetic parameter values. We test the model by comparing predictions made in mutant strains with qualitative experimental observations made in the same strains. Finally, we use the model to predict nutrient-responsive transcription factor activities in a number of mutant strains undergoing complex nutrient shifts.
... The process requires an energy source such as the fermentation of glucose (Taylor, 1949; Davies, Folkes, Gale & Bigger, 1953; Halvorson & Cohen, 1958; Eddy & Indge, 1961), and aerobic conditions (Massin & Lindenberg, 1958). It is temperature dependent and is blocked by such metabolic inhibitors as azide and 2,4-dinitrophenol (Halvorson, Fry & Schwemmin, 1955 ; Halvorson & Cohen, 1958). In several studies amino acid accumulation has been shown to be inhibited by the presence of other amino acids (Taylor, 1949; Halvorson & Cohen, 1958; Massin & Lindenberg, 1958). ...
Article
SUMMARY A study of the accumulation by a brewer's yeast of six sulphur-containing amino acids labelled with sulphur-35 showed several distinct patterns of uptake. L-Methionine was accumulated rapidly and completely from the medium. L-cysteine was taken up slowly, and L-cystine not at all. DL-Ethionine accumulation was incomplete. S-Methyl-L-cysteine and S-ethyl-L-cysteine were taken up rapidly for a short period, and part of the sulphur of these compounds was then released into the medium. Accumulation of the sulphur amino acids was inhibited by certain other amino acids with a close structural relationship to them and when present in high concentration. Cysteine uptake was enhanced by the presence of reduced glutathione. These effects on accumulation were shown to be primarily changes in the transport of the sulphur amino acids into the yeast, although subsequent incorporation into protein may also be affected.
... There was no net synthesis of penicillinase by bacilli of strain 749/c placed in a nitrogen-free medium; under comparable conditions yeast protoplasts formed invertase for several hours at a rapid rate which was not increased by adding a mixture of amino acids (Lampen, 1965). This difference may reflect the relatively large amino acid pool in yeast (Halvorson, Fry & Schwemmin, 1955). The release of penicillinase was relatively rapid at first;Table 2). ...
Article
The quantity of free amino acids within exponentially-growing cells of Staphylococcus aureus is not affected by the osmotic pressure of the external medium, in contrast to that of Escherichia coli B. All the extraction procedures investigated, including most commonly-used techniques, liberate similar quantities of amino acids (of the order of 200–500 μmole/g cells); progress curves of extraction have been determined.Chromatographic examination shows the presence in this internal pool of all amino acids found in the cell protein; major components are proline, glutamic and aspartic acids, isoleucine, methionine and alanine. Concentration gradients exist of most amino acids across the cell surface; these gradients are greatest for proline, glutamic and aspartic acids, isoleucine and methionine.An upper limit has been estimated to the quantity of TCA-soluble peptides which are present in exponentially-growing cells; one dialysable peptide, containing glutamic acid, glycine, alanine and lysine, has been detected in TCA extracts.
Article
Transfer ribonucleic acid (tRNA) that is deficient in methyl groups may be detected in logarithmically growing Saccharomyces cerevisiae. The amount of methyl-deficient tRNA is not constant throughout the logarithmic phase, but is maximal about one generation before the onset of the late growth phase. During this latter phase, the tRNA is fully methylated. The methyl-deficient tRNA is present during a period of high metabolic activity of the cell, characterized by increased RNA and protein content.
Article
1.1. Saccharomyces chevalieri cells grown in complex medium or in synthetic medium containing (NH4)2SO4 as the sole nitrogen source show a reduced capacity to accumulate [14C]proline.2.2. The uptake of proline is greatly enhanced by incubating the cells without further growth in a nitrogen-free medium, with galactose as the sole energy source. The enhancement in uptake capacity by nitrogen starvation is not affected by anaerobic or fully aerobic conditions. Also, the increase in proline pool is without effect.3.3. Cycloheximide, blasticidine-S and sparsomycin inhibit the derepression. Also, the amino acid analogs and 5-methyltryptophan prevent the increase of proline uptake. Benzimidazole, proflavine and ethidium bromide are effective inhibitors of the derepression.4.4. Other amino acids do not show such an increase in accumulation capacity after nitrogen starvation. For some amino acids which show a moderate transport enhancement, such an increase is not abolished by cycloheximide.5.5. Results are discussed in terms of the idea that the biosynthesis of a protein necessary for the transport of proline is repressed during growth of Saccharomyces chevalieri and that derepression occurs when the cells are subjected to nitrogen starvation.
Article
Full-text available
Mitochondrial protein synthesis was studied during release from glucose repression in Saccharomyces cerevisiae cells bearing different mitochondrial genomes. The increase in the rate of the synthesis of mitochondrial translation products was analyzed during respiratory induction. Different kinetic patterns were found for strains having a different structure of mitochondrial mosaic genes, even when the nuclear background was the same. A very limited response of the synthesis of the var1 ribosomal protein to inducing conditions was observed.
Article
In the absence of exogenous energy and nitrogen, resting yeast cells are capable of replenishing both their nucleotide and free amino acid pools. This replenishment phenomenon is the result of intracellular protein and nucleic acid breakdown rather than of cell lysis. Isotopically labeled cells showed turnover rates of 6.6 · 10−3 h−1 and 1.5 · 10−3 h−1 for protein and nucleic acid respectively. Protein and nucleic acid degradation, as well as their synthesis, are energy-requiring reactions.When exogenous energy is available, the degradation products are reutilized for protein and nucleic acid synthesis. Employing 15N-purine-grown cells, it was found that the amino groups of nucleic acid purines can serve as nitrogen reservoirs for limited protein synthesis.
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Article
Sarcina lutea, harvested from peptone media, displays a high rate of endogenous metabolism which may be diminished to a negligible level by aeration of cell suspensions at 37°. During the process, ammonia appears in the supernatant fluid and the free amino acid pool decreases by approximately one half, remaining constant when the metabolism has fallen to negligible proportions; there is no concomitant decrease in either cell carbohydrate or fat.The ultracentrifugal pattern of cell extracts does not change significantly during diminution of endogenous metabolism and the observed oxygen consumption can be accounted for solely by the oxidation of amino acids and peptides within the pool.Endogenous metabolism is restored by the incubation of cell suspensions with casein hydrolysate, glucose plus ammonium salt and glucose alone, in that order of decreasing effectiveness. In each case there is a good correlation between the increase in Q02 and the increase in the free amino acid-pool concentration; the increase of the pool when cells are incubated with glucose alone is attributed to the mobilization of some endogenous peptide or protein material. The pattern of the amino acid pool has been investigated chromatographically during the diminution and restoration of endogenous metabolism. It is concluded that amino acids are the substrates for endogenous metabolism.Incubation with glucose produces extensive intracellular carbohydrate deposition whereas incubation with casein hydrolysate is without effect on the carbohydrate content of the cells.High levels of endogenous metabolism and the oxidation of exogenous glucose are mutually inhibitory.
IntroductionThe Induction PhenomenonCurrent Model of Protein SynthesisNucleic Acids and Enzyme InductionSpeculations on the Mechanism of Inducer ActionSummary and Conclusions
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The amino acid pool of yeast cells, Saccharomyces cerevisiae, incubated with galactose remains at a constant level for 100 minutes. This is 30 minutes beyond the time at which the oxidative phase of the induced-enzyme formation begins. Washed yeast cells, the pools of which have been depleted 60 per cent by incubation with glucose, do not replenish their pools as do washed cells incubated without a substrate. These facts indicate that the induced enzymes are formed at least partially from pool-replenishing amino acids. The time of onset of pool depletion is the time at which the aerobic fermentation phase of induced-enzyme formation begins for cells incubated with galactose. With 0.1 per cent galactose the respiratory phase begins at 100 minutes but no aerobic fermentation nor pool depletion occurs. The rates of respiration and aerobic fermentation are constant for four glucose concentrations from 0.1 to 1.0 per cent. The amount of aerobicfermentation is proportional to the initial concentration of glucose. Amino acid pool depletion occurs for all concentrations but depletion ceases and is followed by pool replenishment after aerobic fermentation is complete. Ultraviolet radiations, which delay the appearance of the respiratory phase of induced-enzyme formation, completely eliminate both the appearance of aerobic fermentation and pool depletion. The results indicate an intimate association between aerobic fermentation and amino acid pool depletion.
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Methionine auxotrophs of Saccharomyces cerevisiae continue to synthesize ribonucleic acid (RNA) after methionine withdrawal. The newly synthesized transfer RNA (tRNA) is methyl-deficient in some strains, but not in all. Whether such tRNA will accumulate depends on the position of the block in the methionine pathway that is carried by the mutant strain. Free methionine rapidly decreases in the intracellular pool of all strains after its removal from the medium. Certain metabolites derived from methionine are removed from the pool relatively slowly after methionine withdrawal. Notable among these is S-adenosylhomocysteine, which is depleted less rapidly from those strains that accumulate methyl-deficient tRNA than from others. S-adenosylhomocysteine is a potent inhibitor of tRNA-methylating enzymes in vitro.
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In Streptomyces hydrogenans an active transport of glutamate takes place. The accumulation is highly sensitive to metabolic inhibition. The distribution ratio varies with the extracellular substrate concentration. This ratio was found to be 10 to an extracellular concentration of 3 mM. It is concluded that glutamate is transported via two systems which differ widely in their kinetic parameters. Our results indicate that most of the intracellular glutamate is in the free state and not bound to distinct sites of the cell. The amount of intracellular glutamate is about 160 μmoles per g dry weight, and this pool can be drastically reduced by cold shock treatment. The replenishment of the pool is complete over a wide range of extracellular glutamate concentrations, and it seems that the capacity of the specific system is effective enough to build up a nearly normal glutamate pool. The influx of glutamate is influenced by the composition of the endogenous pool.
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The properties of an amino acid accumulating system were investigated in Penicillium griseofulvum Dierckx by following the uptake of l-leucine (and other amino acids) by mycelium in the absence of external glucose. In these conditions 98% of the leucine accumulated was shown to be in unbound form in the cell. The kinetics of uptake at 25 °C may be described by a Michaelis—Menten relation with an apparent Km of 2·22 × 10−3m. Uptake of leucine by the mycelium was abolished by several metabolic inhibitors, and the amount accumulated was markedly dependent on the pH of the medium, on the age of the mycelium, and on the source of nitrogen on which the mycelium had previously been growing. Studies of simultaneous uptake of several amino acids suggested the presence of more than one mechanism of uptake with broad but differing specificities.
Chapter
Auch der Zustand der nichtwachsenden, aber lebenden und aktiven Zelle ist, wie wir wissen, dynamisch charakterisiert, als Gleichgewichtszustand, in dem der dauernd ablaufende Stoffabbau durch eine Synthese gleichen Umfanges bilanzmäßig kompensiert ist (Rittenberg und Mitarbeiter 1939, Vickery und Mitarbeiter 1939, Hevesy und Mitarbeiter 1940, Schoenheimer 1942, MacVicar und Burris 1948, Mazia und Prescott 1955 u. a.). Überwiegen die Synthesen die abbauenden Vorgänge, so arbeitet die Zelle mit Stoffgewinn, sie wächst. Diese Art des Wachstums, die wir, soweit wir nur die Zunahme der plasmatischen Zellbestandteile ins Auge fassesn, als Plasmawachstum bezeichnen, ist die fundamentale, die gewöhnlich auch den anderen Wachstumsformen, dem Teilungswachstum und dem Streckungswachstum, vorauszugehen hat, diese auch zum Teil noch begleitet.
Chapter
In respiration all plants exchange gases with the surrounding medium, and with the exception of a few microorganisms this exchange consists of an absorption of oxygen by the plant from the medium and an evolution of carbon dioxide from the plant into the medium. With most vascular plants the medium is a double one, air and soil, exceptions being found in a few completely aerial plants and in water plants which may or may not be rooted in soil and which may be completely or only partially submerged. With most of the Bryophyta the bulk of the gaseous exchange takes place between the plant and air, the rhizoids attaching the plants to the soil forming only a very small portion of the plant. Apart from a comparatively small number of terrestrial forms the algae live in an aqueous medium which may be a weaker or stronger solution, mainly of inorganic salts, according to whether the plants live in fresh or salt water. The media with which gaseous exchange takes place are thus air, soil, fresh water and salt water, according to the species.
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This chapter discusses the theories of enzyme adaptation in microorganisms. The theories are grouped into two categories: those that are specifically concerned with enzyme adaptation, and those that are based on more generalized cell models and can be used, in principle at any rate, to account for other phenomena, such as cell growth. Enzyme adaptation is an increase in the activity of a specific enzyme brought about by the presence of a specific substance, usually the substrate, occurring without any change in the genotype. A basic phenomenon of enzyme adaptation is presented. If organisms are incubated with the substrate, or some chemically similar compound, increased enzyme activity occurs quite rapidly, often within a few minutes, and this occurs due to the synthesis of specific enzymes. A source of energy is invariably necessary, and if the inducer itself cannot provide energy, some other suitable donor must be provided.
Article
The formation of isobutanol and 3-methylbutanol during alcoholic fermentation by brewer's yeast has been studied by adding uniformly 14C-labelled valine and leucine to the complex amino acid mixture which is used as nitrogen source. In the presence of 14C-leucine, only 3-methylbutanol becomes radioactive whereas, in the presence of 14C-valine, both isobutanol and 3-methylbutanol acquire label. The specific radioactivity of these compounds is, in all cases, inferior to that of the parent amino acids showing that a part of these higher alcohols is always formed by the synthetic functions of the yeast. The results suggest that the regulating effect of the valine and leucine level of the medium on the synthesis of these amino acids in yeast cells is rather limited. They furthermore indicate that intact assimilation of amino acids is not the only mode of their utilization, even in media with excessive levels of amino acids. An attempt is made to explain the dependence of the anabolic and catabolic modes of higher alcohol formation upon the nitrogenous nutrient level of the medium. The presence of radioactive isoleucine as impurity in the 14C-leucine used has allowed an approximative calculation of the formation of 2-methylbutanol from isoleucine which is very similar to the formation of isobutanol from valine.
Article
Meiosis is induced in yeast by a sequence of events starting with the disappearance of glucose from the growth medium. Preparation for meiosis during the period of the premeiotic mitoses involves: (1) Development of the ability to metabolize acetate; and (2) establishment and maintenance of sufficiently high concentrations of RNA and protein in the cell to support an extensive biosynthetic activity. A new regulation model is proposed on the basis of the results obtained.
Article
Induction of meiosis in yeast is a complex process starting during the period of premeiotic mitoses. Enlargement of the nucleus is the earliest sign of the actual onset of meiosis. Parallel with this development is an increase of about 10% in the protein content of the cell. Somewhat later there is a sharp rise in cell mass and cell volume. There is only one period of high metabolic activity during sporogenesis and this occurs early during the process.
Article
Der Zusatz von Glukose in Gegenwart von Luft zu « resting cellsder Hefe Saccharomyces cerevisiae, welche in Anaerobiose mit Glukose als Kohlenstoffquelle kultiviert wurde, bewirkt : — die Synthese der Cytochrome a + a3, b, c1 und c und die Zunahme der Cytochrom c - Oxydase und der Succinat-Cytochrom c-Reductase(Atmungsadaptation) ; die Geschwindigkeit dieser Synthesen hängt von der Anfangskonzentration der Glukose im Adaptationsmedium (« contre effet Pasteur ) ab ; sie ist desto rascher je weniger Glukose vorhanden ist(Slonimski [4]) ;— das rasche Verschwinden der δALA-Synthetase (weniger als eine Stunde), welche im Laufe der ganzen während 9 Stunden bei 25° gemessenen Atmungsadaptation nicht mehr gefunden wird ; das Vorhandensein starker Mengen von freiem δALA zur Zeit O der Adaptation erlaubt jedoch die Bildung der gesamten Cytochrome der Atmungskette zu erklären ;— das rasche Verschwinden eines (oder mehrerer) Enzyme der Synthesekette des Protohäms, welche zwischen dem Porphobilinogen und dem Protoporphyrin liegen ; das verschwundene Enzym, welches dem Komplex PBG-Desaminase-Uroporphyrinogen III-Cosynthetase zu entsprechen scheint, wird de novo synthetisiert, nachdem die Glukose aus dem Adaptationsmedium verschwunden ist. Der Mechanismus des Verschwindens dieser Enzymaktivitäten scheint nicht von der Synthese von einer oder mehrerer dieser Aktivitäten in Gegenwart von Glukose hemmenden Proteinen abzuhängen ; dieses Verschwinden wird auch beobachtet, wenn die « resting cellsin Gegenwart von Glukose und in Abwesenheit von Sauerstoff inkubiert werden. Gleiche Ergebnisse werden bei den « resting cellsder in Aerobiose mit Glukose als Kohlenstoffquelle kultivierten Hefe gefunden.
Article
In Streptomyces hydrogenans an active transport of glutamate takes place. The accumulation is highly sensitive to metabolic inhibition. The distribution ratio varies with the extracellular substrate concentration. This ratio was found to be 10 at an extracellular concentration of 3 mM. It is concluded that glutamate is transported via two systems which differ widely in their kinetic parameters. Our results indicate that most of the intracellular glutamate is in the free state and not bound to distinct sites of the cell. The amount of intracellular glutamate is about 160 μmoles per g dry weight, and this pool can be drastically reduced by cold shock treatment. The replenishment of the pool is complete over a wide range of extracellular glutamate concentrations, and it seems that the capacity of the specific system is effective enough to build up a nearly normal glutamate pool. The influx of glutamate is influenced by the composition of the endogenous pool.
Article
In vorhergehenden experimentellen Arbeiten diente der sogenannte produktive Eiweißnutzwert (PEW=N-Zuwachs des Organismus/N-Zufuhr)tät. Ergänzend wird die Anwendung dieses Bewertungssystems in vorliegender Veröffentlichung ausführlich begründet. Gegen die Einbeziehung des endogenen Harn-N (EHN) in die Formel zur Berechnung der biologischen Wertigkeit sprechen u. a. die neueren Vorstellungen vom dynamischen Verlauf des Eiweißstoffwechsels. Die Erfordernisse der Ernährungspraxis lassen es außerdem zweckmäßig erscheinen, das Verhältnis von Eiweißaufwand im Futter zum Eiweißertrag im Tier als Bewertungsmaßstab zu benutzen.
Article
A study was made of the effect of ultraviolet radiations on leakage of ; amino acids from yeast cells and on the amino acid pools of these cells. Results ; are tabulated. The significance ot the findings is discussed. Leakage of amino ; acids from yeast cells following ultraviolet radiation is concluded to be a ; selective process influenced by the metabolic activity of the cell. Membrane ; damage probably occurs but it alone cannot account for the observed facts. ; (C.H.);
Article
Although the amino acid nutrition of fungi has received considerable attention, less study has been made of amino acid transport into fungal cells. Cochrane (1958), in a paragraph in his book dealing with the transport of amino acids into the cell stated, ‘the main purpose of this section must be to call attention to a problem which has only been tentatively explored in the fungi’.This essay considers the evolution of transport systems, properties of amino acid transport systems and the possible mechanisms of their operation. An attempt is made to correlate physiological and ecological data regarding amino acids and fungi, and to consider whether these amino acid transport systems are important functional processes in a natural environment. In this connexion it is necessary to consider aspects of nitrogen nutrition.
Article
1.(1.) Propionaldehyde, a substrate capable of activating electron transfer and oxidative phosphorylation, stimulated the concentrative uptake (entrance and accumulation) of l-[14C]leucine by starved Saccharomyces ellipsoideus. Under adequate experimental conditions the ratio of l-[14C]leucine uptake to oxygen uptake was higher with propionaldehyde than with d-glucose or endogenous substrates as energy sources.2.(2) With d-glucose as energy source, antimycin significantly inhibited l-[14C]leucine entrance and accumulation but the inhibition was less than that of respiration. With propionaldehyde as the energy source, cycloheximide did not affect l-[14C]leucine uptake despite the almost complete inhibition of amino acid incorporation into the cell protein.3.(3) Preincubation of starved yeast with l-glucose or propionaldehyde before l-[14C]leucine addition significantly increased the amino acid entrance values. The effect of energization on amino acid entrance increased with the time the yeast was preincubated with the oxidizable substrates. Energization of a S. ellipsoideus rho− mutant with d-glucose, caused less l-[14C]leucine entrance than with the wild type yeast.4.(4) Addition of 2,4-dinitrophenol to the d-glucose or propionaldehyde-energized S. ellipsoideus brought about a significant inhibition of l-[14C]leucine entrance, but the d-glucose-energized yeast was much less sensitive to the uncoupler than its propionaldehyde-energized counterpart. 2,4-Dinitrophenol also inhibited l-[14C]leucine entrance in the d-glucose-energized rho− mutant, to the same extent as with the d-glucose-energized wild type yeast. Preincubation of yeast with 2,4-dinitrophenol prevented to about the same extent the energization of l-[14C]leucine transport either by d-glucose or by propionaldehyde.5.(5) The kinetics of l-[14C]leucine transport as a function of amino acid concentration indicate the existence of two apparently distinct transport systems, namely, one with high affinity and low transport activity (System A) and the other with low affinity and high transport activity (System B). The kinetic parameters KT and V characterizing these systems were dependent on the energization state of the yeast cells since KTA (starved) < KTA (energized); KTB (starved) >KTB (energized), while V (starved) < V (energized), (for both systems). Systems A and B could be further differentiated by the effect of pH and temperature.6.(6) Ammonium ions significantly inhibited l-[14C]leucine entrance in d-glucose energized yeast when added simultaneously with d-glucose, but stimulated the very early entrance when added after energization had occurred. With the glucose-energized rho− mutant, ammonium ions increased l-[14C]leucine entrance irrespective of the time addition.7.(7) Preincubation of yeast with cyclic AMP caused an increased rate of l-[14C]leucine entrance. The effect (a) was related to the nucleotide concentration, and (b) was higher with 1.0 mM than with 0.1 mM l-[14C]leucine. This difference indicates that l-[14C]leucine transport System B was the more responsive to cyclic AMP.
Article
(1) Substrates capable of activating mitochondrial electron transfer and oxidative phosphorylation, namely, pyruvate, acetate, propionaldehyde and butanol, stimulated the concentrative uptake (transport and accumulation) of L-[14-C]leucine by Saccharomyces cerevisiae (wild type strain 207, starved cells). Under adequate experimental conditions, the L-[14-C]leucine uptake versus the oxygen uptake ratio was almost the same with either pyruvate, acetate or D-glucose as energy sources. Substrate oxidation also increased L-[14-C]leucine incorporation into the cell protein. (2) With S. cerevisiae D261 and D247-2 and propionaldehyde as an energy source, or with strain 207 and glucose as energy source, 2,4-dinitrophenol (50 muM) inhibited L-[14-C]leucine uptake, the inhibition being accompanied by stimulation of respiration. With S. cerevisiae 207 and propionaldehyde as energy source, 2,4-dinitrophenol inhibited both respiration and L-[14-C]leucine uptake, but with respiration being less affected than uptake. Displacement of accumulated L-[14-C]leucine was also inhibited by 2,4-dinitrophenol. (3) In the presence of glucose, and for relatively brief incubation periods, anaerobically grown cells of S. cerevisiae 207 and of a p-minus "petite" mutant of this strain incorporated L-[14-C]leucine with less efficiency than the original wild type strain 207, grown aerobically. With D-glucose as energy source, 2,4-dinitrophenol and iodoacetate inhibited alike L-[14-C]leucine uptake by the respiration competent cells. (4) It is postulated that in respiration-competent yeasts, the mitochondrion contributes to 6-[14-C]leucine uptake by supplying high-energy compounds required for amino acid transport and accumulation. Conversely, the promitochondrion in the anaerobically grown yeast, or the modified mitochondrion in the respiratory deficient mutant, competes for high energy compounds generated by glycolysis in the cytosol.
Article
1.1. A polyploid series from haploid through tetraploid in yeasts has been analyzed for a number of cellular characters including dry weight, desoxyribonucleic acid (DNA), ribonucleic acid (RNA), and metaphosphate content.2.2. The DNA content per cell is found consistent with an integral ratio in the polyploid series, and this character has proved most reliable of those studied in estimating ploidy.3.3. Dry weight, RNA, and metaphosphate content per cell are also ploidy-dependent but vary over a wider experimental range.4.4. The reliable estimation of ploidy in yeast provides a critical tool for the analysis of irregular segregation in the yeasts.
Article
This chapter discusses the assimilation of amino acids by gram-positive bacteria. The growth of the bacterial cell involves, and is the result of, synthesis of all its components. Many bacteria can synthesize protein from ammonia and a carbon source, such as glucose. “Biochemical mutants” of molds and bacteria have indicated the probable biosynthetic pathways and individual steps in pathways that have been studied with cell-free extracts of microorganisms. Species of bacteria that are unable to synthesize amino acids, assimilates the preformed substances, to form suitable material for the study. Experimental correlations among the various processes are observed, and certain stages in the processes are found to be sensitive to antibiotics and growth inhibitors.
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