A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Compartmentalization of metabolism of spatially-delimiting amino acid pools of yeast cells

The spatially different amino acid pools (i.e. cytoplasmic, vacuolar and mitochondrial) of yeast cells are metabolically compartmentalized. The accumulation of amino acids in these pools occurs at different rates; the highest rates are observed for glutamate and alanine. The former is predominantly accumulated in the cytoplasm, the latter--in the vacuoles. The renewal rates of the amino acid pools are also different. Each of them contains at least two subpools, readily convertible and relatively stable ones. The readily convertible subpools of the cytoplasmic and mitochondrial pools predominantly contain glutamate, aspartate, valine and alanine; that of the vacuolar pool--alanine. The bulk of the readily convertible alanine subpool (67%) is localized in the vacuoles, that of glutamate and aspartate (85 and 68%, respectively)--in the cytoplasm.     

Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae.

By using the Cu2+ method (Y. Ohsumi, K. Kitamoto, and Y. Anraku, J. Bacteriol. 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined. Histidine and lysine in the medium expanded the vacuolar pool extremely. Glutamate also accumulated in the cells, but mainly in the cytosol. The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool. Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM. This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation. Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate. The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.     

Effect of biotin on ribonucleic acid synthesis.

A single injection of biotin to biotin-deficient rats produces a two-fold increase in the incorporation, both in vivo and in vitro of precursors into nucleic acids as early as 2 h after the biotin treatment. The specific activity of the precursor pool is not affected by biotin. Analysis of the polysome profile at various times following biotin treatment and a kinetic study of the effect of excess poly(U) on the incorporation of phenylalanine by cell-free amino acid incorporation experiments indicate a marked decrease in messenger-free ribosomes in rat liver after biotin administration.     

Biotin uptake, utilization, and efflux in normal and biotin-deficient rat hepatocytes.

Biotin uptake, utilization, and efflux were studied in normal and biotin-deficient cultured rat hepatocytes. Biotin-deficient cells accumulate about 16-fold more biotin than do normal cells when incubated with a physiological concentration of biotin for 24 h. This difference is due to the greater amount of protein-bound biotin relative to free biotin in biotin-deficient hepatocytes, and is attributable to the presence of more apocarboxylases in deficient cells. The rate of biotin uptake and the rate of activation of the carboxylases, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase, are proportional to the concentration of exogenous biotin. Increases in carboxylase activities are proportional to the concentration of biotin only at exogenous biotin concentrations of less than 410 nM. Concentrations of 410 nM or more biotin increase carboxylase activities to normal or near normal. Biocytin inhibits biotin uptake at very high concentrations, whereas desthiobiotin and lipoic acid have no effect. Biocytin in the medium results in carboxylase activation either intracellularly or extracellularly by conversion to biotin by biotinidase. Investigation of the efflux of biotin from normal and biotin-deficient cells preincubated with the vitamin showed greater retention of biotin by biotin-deficient cells than by normal cells over 24 h. Retention of free biotin is similar in biotin-deficient and normal cells. The greater amount of biotin retained by biotin-deficient cells is accounted for by the greater amount of bound biotin in these cells. These results suggest that the free and bound biotin pools are independently regulated. The ready loss of free biotin from these cells has implications for the treatment of inherited, biotin-responsive carboxylase deficiencies.     

Impact of UV radiation on the production patterns and composition of dissolved free and combined amino acids in marine phytoplankton

Using the ¹³C tracer technique in conjunction with gas chromatographic-massspectro-metric (GC-MS) techniques, we examined the patternsof synthesis and the composition of dissolved free and combinedamino acids within phytoplankton photosynthesizing in the presenceand absence of natural solar ultraviolet radiation (UVR). Atlevels that still permitted the uptake of carbon assimilationinto the cells, UVR caused a marked decline in the overall rateof carbon incorporated into amino acids and a reduction in thepool size of total cellular amino acids (TCAA). In contrast,absolute concentrations of amino acids within the intracellulardissolved free amino acid (INDFAA) pool (measured using an aminoacid analyzer) were higher in the presence of UVR. An examinationof the production patterns and composition of amino acids constitutingthe INDFAA and TCAA pools revealed a marked diminution in thesynthesis and accumulation of alanine and valine in the presenceof UVR. On the other hand, the rates of synthesis and concentrationsof glutamic acid (glutamic acid + glutamine) in the INDFAA andTCAA pools of phytoplankton were higher in samples exposed toUVR. These changes are discussed with reference to the knowneffects of UVR on nitrogen and carbon assimilation within phytoplankton.     

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.     

Morphogenesis and free amino acid composition of the ascomycete Sphaerostilbe repens as influenced by nitrogen and calcium

Abstract Sphaerostilbe repens utilizes nitrate and ammonium as nitrogen sources. Differentiation of mycelium into rhizomorphs and coremia was reduced in the presence of nitrate and completely inhibited in the absence of calcium. The most abundant free amino acids were, in decreasing order: alanine, glutamine, glutatomic acid, serine, aspartic acid, γ-aminobutyric acid, arginine and threonine. These compounds represented 90% of the total amino acid pool.
The free amino acid composition did not vary with cultural conditions although concentrations of individual amino acids differed. In ammonium-grown cells, γ-aminobutyric acid increased in concentration and glutamate, aspartate and alanine decreased. Calcium-deficient media reduced amino acid concentrations, especially of arginine and ornithine. Amino acid contents increased during the growth period and were higher in rhizomorphs than in vegetative mycelia.     

Nitrogen Starvation-Induced Changes in Amino Acid and Free Ammonium Pools in <Emphasis Type="Italic">Schizophyllum commune</Emphasis> Colonies

Wood-decay fungi depend upon recycling of nitrogen-containing molecules to maintain growth in nitrogen-deficient environments. One of the pools that can support growth in these organisms is the pool of free amino acids. The free amino acid (AA) composition of Schizophyllum commune mycelium grown on the surface of nitrogen-rich (M = 6.6 mM L-asparagine) and nitrogen-poor medium (M01 = 0.06 mM L-asparagine) has been examined: When mycelium is grown on M, alanine, glutamate, and asparagine account for almost 2/3 of the amino acid pool. The free amino acid concentration is reduced by 75% for mycelium grown on the M01 medium, with alanine and glutamate predominating. In addition, free NH₄⁺ increases by 60% in nitrogen-deprived mycelia. Except for asparagine, which is absorbed by the apices, the concentration of all free amino acids is higher in the centers of M-grown, 4-day-old mycelia than in the apices. Hyphae grown to exponential growth on M and transferred to M01 for 12 h show greater free amino acid and NH₄⁺ concentrations in the apices, most likely indicating increased translocation to the apices.     

Glutamine requirement for aerial mycelium growth in Neurospora crassa

Five amino acids are accumulated during vegetative growth of Neurospora crassa, particularly.during the prestationary growth phase. Alanine, glutamine, glutamate, arginine and ornithine.comprised over 80% of the total amino acid pool in the mycelium. Amino acid pools of different amino acid auxotrophs were followed during the partial transformation of a mycelial mat into an aerial mycelium. The mycelial mat under starvation and in direct contact with air rapidly formed aerial mycelium, which produced thereafter a burst of conidia. During this process,glutamine and alanine in the mycelial mat were consumed more rapidly than other amino acids;in the growing aerial mycelium, glutamate and glutamine were particularly accumulated. Of the amino acids that were initially accumulated in the mycelial mat, only a high glutamine pool was required for aerial mycelium growth induced by starvation. This requirement for glutamine could not be satisfied by a mixture of the amino compounds that are synthesized via glutamine amidotransferase reactions. It is proposed that glutamine serves as a nitrogen carrier from the mycelial mat to the growing aerial mycelium.     

Aerobic Fermentation and the Depletion of the Amino Acid Pool in Yeast Cells

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.     

Effect of 24-hour starvation on amino acid pool composition and enzyme activities of rat brown adipose tissue

The effect of 24-hr starvation on the amino acid pool composition and its concentration ratios with respect to blood and plasma as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase have been studied in rat brown adipose tissue. Starvation induced a considerable decrease of pool amino acid concentration. Alanine and taurine were the amino acids in which the decrease was more marked. Small changes were observed in the activities of the enzymes studied, with decreases only in glutamate dehydrogenase and adenylate deaminase. These changes agree with a decrease in amino acid utilization in this tissue induced by starvation.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

The establishment of essential and metabolically derived amino acid profiles in developing chick embryo organs

The accumulation of certain essential and metabolically derived amino acids in the free amino acid pools of three excitable tissues has been studied in the chick embryo. Valine together with leucine are at the onset present in the yolk at higher concentrations than any of the other essential amino acids. By 15 days all the amino acids studied have accumulated in the embryonic pools at a higher rate than valine, although certain amino acids, such as phenylalanine or methionine, always remain at lower relative concentrations than valine. This reflects their low supply in the yolk, rather than a more rapid rate of disappearance (utilization). During early embryogenesis (E₂–E₄), tissues preferentially concentrate glutamic acid, besides taurine and phosphoethanolamine (6). The next distinct stage of development (E₄–E₇) is marked in the brain by a gradual rise in glutamic acid, glutamine and aspartic acid; the same three amino, acids do not demonstrate a further increase in the pool of the heart, while in the whole eye the amino acid profile begins to resemble the blood. Leucine in all three tissues declines rapidly, to reach isoleucine levels by day 7 of development; tyrosine increases slowly in apparent reciprocity to an equally gradual phenylalanine decrease. Into the second week of embryo growth (E₇–E₁₅), GABA appears in the mesencephalon (E₇) and the eye (E₉–E₁₀). In the mesencephalon, the free amino acid pool composition exhibits a rather sudden increase of most metabolically derived amino acids. Glutamic acid and glutamine in the brain increase in parallel; the rate of GABA and aspartic acid accumulation is slower, and for GABA stabilizes on day 14, as does glutamine. In the eye, by day 15, GABA levels are more closely aligned with the aspartic acid content. Finally, throughout embryogenesis serine fluctuations in blood and tissues are in parallel with those of threonine, and different from glycine or alanine which also change in tandem.     

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-3H]aspartate

Kainic acid, a powerful neuroexcitant and neurotoxin, stimulates the release of naturally occurring excitatory amino acids, l-glutamate and l-aspartate, from hippocampal synaptosomes. The release stimulation affects in a similar way both the general pool of the two amino acids and the fraction of l-glutamate and l-aspartate, newly-synthetized from precursors or recently accumulated through the high-affinity uptake mechanism. Kainic acid exerts its stimulatory action on the basal release of the two amino acids as well as on the high K⁺-stimulated release of l-glutamate. Kainic acid has, however, different effects on the release of exogenously accumulated [d-³H]aspartate. In particular, the high K⁺-stimulated release of this false transmitter is strongly inhibited by 1 mM kainic acid. The present data confirm the presynaptic action of kainic acid on the general as well as on the recently-formed pools of naturally occurring excitatory amino acids. At the same time, our results suggest that [d-³H]aspartate is not a reliable substitute for l-glutamate and l-aspartate, in release studies and that the radioactivity released after preloading with [d-³H]aspartate does not necessarily reflect the release of naturally occurring excitatory amino acids.     

Salt Stress Control of Intracellular Solutes in Streptomycetes Indigenous to Saline Soils

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (−3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K⁺ salts sharply increased.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid pool composition of the basidiomycete Coprinus cinereus

The leaf-litter fungus Coprinus cinereus maintains a pool of free amino acid in its mycelium. When the organism is grown under conditions of high nitrogen availability with 13.2 mmol.L-1 L-asparagine as the nitrogen source, the primary constituents of this pool are glutamine, alanine, and glutamic acid. Together these 3 amino acids comprise approximately 70% of the pool. Nitrogen deprivation reduces the size of the free amino acid pool by 75%, and neither a high concentration of ammonium nor a protein nitrogen source support a similar pool size as L-asparagine. Nitrogen deprivation also reduces the concentration of glutamine to the pool while increasing glutamate. Concomitant with this shift is a marked increase in mycelial ammonium.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.