The metabolic properties of acid soluble polyphosphates in Saccharomyces cerevisiae

Summary Tripolyphosphate was found to be the predominant species of soluble polyphosphate in yeast. Evidence is presented which shows that under normal growth conditions tripolyphosphate had little or no turnover. The amounts of the various polyphosphates decreased as the chain length increased. Tetrapolyphosphate was shown to be synthesized more rapidly than tripolyphosphate. These observations suggest that short chain polyphosphates arise by degradation of longer chain length polyphosphates with tripolyphosphate the ultimate degradation product.During nitrogen starvation, the normal accumulation of tripolyphosphate rapidly ceased even though the cells continued normal growth for at least two hours. After the addition of L-amino acids or (NH₄)₂SO₄ to nitrogen starved cells, there was a dramatic increase in the accumulation of tripolyphosphate and tetrapolyphosphate which occurred at the same time as the increase in growth rate. Implications of this result are discussed in terms of possible functions of polyphosphate.     

Effect of threonine, cystathionine, and the branched-chain amino acids on the metabolism of Zygosaccharomyces rouxii*

Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of alpha-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of alpha-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of alpha-ketobutyrate but that the alpha-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of alpha-ketobutyrate accumulation.     

Effect of surface-active agents (tween-21) on indices of energy metabolism in oleandomycin producers

The specific growth rate of Streptomyces antibioticus, a producer of oleandomycin, and the specific rate of the antibiotic accumulation in the culture medium during fermentation were investigated. On the basis of the results obtained the fermentation period was divided into 7 phases of development. The culture treated with the surfactant (Tween-21) is characterized by a higher specific growth rate during the whole fermentation and a higher specific rate of the antibiotic accumulation at the stage of the highest production as compared to the control. The ATP content, the value of the adenylate energy charge and the contents of high-molecular weight polyphosphates in the mycelium were examined. In the phase of the intensive growth St. antibioticus was characterized by a higher ATP level and a higher energy charge. More active accumulation of polyphosphates was observed in the late intensive growth phase. It was also found that after the treatment of the culture with Tween-21 it utilized polyphosphates more actively during the antibiotic biosynthesis.     

Amino acid limited growth of starter cultures in milk

The specific growth rates of several Streptococcus cremoris strains were 10–40% lower in milk than in other growth in media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maximum specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (K_S) for casein, leucine and glutamate existed between the strains.     

Amino acids as main substrates for sulfate-reducing bacteria in surface sediment of a eutrophic bay

The inner part of Tokyo Bay, Japan, is highly eutrophicated as shown by the frequent occurrence of red tide. The bottom water is anoxic during warm seasons especially at artificially dredged sites. In the sediment slurries prepared from surface sediment samples collected from the dredged sites, substrate addition stimulated the consumption of sulfate during anaerobic incubation. Of the substrates added, the seston composed mainly of diatom stimulated consumption more than lactate and acetate. Its effect was nearly equal to that of casamino acids. Casamino acids and some amino acids also accelerated the rate of sulfate reduction measured by the tracer method in sediment samples more than lactate or acetate. Anaerobic incubation of the sediment slurry amended with casamino acids showed that the consumption of amino acids was retarded by the addition of molybdate (final concentration; 20 mM). In the slurry amended with only molybdate, glutamate was accumulated distinctively and linearly with time. Its accumulation rate in molar base was comparable to the rate of sulfate reduction. These results suggested that amino acids were the main substrates for sulfate-reducing bacteria (SRB) in the sediment. The MPN values of SRB in these sediment samples were often higher with the enumeration medium containing casamino acids instead of lactate. Furthermore, during a week incubation of sediment slurries amended with substrates, casamino acids and seston more greatly stimulated the growth of SRB enumerated by both media than lactate.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

Basic amino acids and inorganic polyphosphates in Neurospora crassa: independent regulation of vacuolar pools

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

The influence of electrical stimulation in vitro on protein synthesis and other metabolic parameters of rat extensor digitorum longus muscle

1. Apparatus is described in which rat extensor digitorum longus muscle can be incubated in buffer under conditions of light tension and be subject to contractures induced by electrical stimulation in vitro. Under these conditions the tissue retains its weight, its content of potassium and size of the extracellular space at values similar to those in vivo. 2. Though uptake of glucose was enhanced on addition of insulin, there was little increase in glucose consumption on stimulation. Breakdown of glycogen and enhancement of lactate output were found on stimulation. 3. Incorporation into protein of several labelled amino acids was diminished during stimulation. Accumulation of [(14)C]leucine was enhanced whereas that of glycine was decreased. 4. There were no very consistent changes in the content of free unlabelled amino acids during incubation with or without stimulation. Comparison of actual amino acid concentrations in tissue and incubation mixture with accumulation of (14)C-labelled amino acid indicated that full equilibration of the cell pool of amino amino acids with the medium is slow. 5. Substantial oxidation of several (14)C-labelled acids was observed. 6. The ATP content of the tissue declined a little during incubation and somewhat faster after a period of stimulation. 7. The results are discussed in relation to the way in which exercise can induce muscle hypertrophy.     

Unsuitable Availability of Nutrients in Germinating Bean Embryos Exposed to Copper Excess

The influence of copper excess on germination rate, growth, minerals, carbohydrates, and amino acids supply in embryonic axis of bean seed was investigated. Compared to the control, Cu treatment caused a reduction in germination percent, embryo length, and accumulation of Ca, Fe, K, Mn, Zn, total soluble sugars, glucose, fructose, sucrose, and amino acids. Moreover, the nutrient concentrations, as well as the electrical conductivity were determined in the germination medium to quantify the extent of solute leakage. Such nutrients were lost in the imbibition medium at the expense of suitable mobilization to the growing embryonic axis. This was associated with an enhancement in accumulation of malondialdehyde, major product of lipoperoxidation process which can be due to the stimulation in lipoxygenase activity in Cu-poisoned tissues.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Stimulating action of polyphosphates on peptide formation from glycine and phenylalanine amides under abiogenic conditions

Synthesis of peptides during polymerization of GlyNH2 and PheNH2 has been demonstrated by means of gel-chromatography and thin-layer chromatography. The optima of pH and temperature have been estimated for the reaction. Grem's salt, tripolyphosphate and pyrophosphate were shown to cause the stimulatory effect on the peptide formation, but there was no correlation between the yield of the peptides and the hydrolysis of the polyphosphates. On the basis of the experimental data, it has been concluded that hydrolysis of polyphosphates is not an energy source for the polymerization of GlyNH2 and PheNH2. Therefore, polyphosphates cause catalytic effect on the peptide synthesis from the amides of amino acids in homogeneous medium.     

Alleviation of Changes in Protein Metabolism in NaCl-Stressed Wheat Seedlings by Thiamine

NaCl-stress induced a pronounced suppression in growth of wheat seedlings. The most abundant amino acids (cysteine, arginine, methionine) constituting about 55 % of total free amino acid content in control wheat were reduced in 100 mM NaCl-treated plants. However, valine, isoleucine, aspartic acid and proline accumulated in response to NaCl stress and NaCl-treated wheat seedlings showed 1.6 fold increase in total free amino acids compared to the control. Addition of 2 [micro ]M thiamine alleviated the effects of NaCl on the amino acid composition and the amount of total free amino acids decreased to that in the control. Content of 26 kDa protein increased in NaCl-treated plants, stimulation was more pronounced in roots than in shoots. In contrast, the contents of 13 and 20 kDa proteins decreased. After addition of thiamine, the 24 kDa protein, which disappeared with NaCl treatment, has been initiated again. Moreover, thiamine treatment stimulated the accumulation of the 20 kDa protein.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Amino acid-dependent increase in hepatic system N activity is linked to cell swelling

Treatment of cultured rat hepatocytes with certain amino acids stimulates the activity of the System N transporter. The present report investigates the mechanism by which the stimulatory amino acids elicit their effect. Activation of System N-mediated transport by amino acids is rapid, cycloheximide-insensitive, and involves neither trans-stimulation nor recruitment of additional carriers to the plasma membrane. In addition, the activation is Na(+)-dependent, supporting the related observation that the most effective stimulatory amino acids are substrates of Na(+)-dependent transport Systems A, ASC, and N whereas substrates of Na(+)-independent System L and non-amino acid metabolites are ineffective. The data suggest that active accumulation of amino acids via Na(+)-dependent carriers is necessary for the activation to occur. The amino acid-dependent stimulation is blocked in a concentration-dependent manner by increasing extracellular K+. Treatment of hepatocytes with an amino acid such as asparagine causes cell swelling and stimulation of System N activity; both of these effects are reduced by hypertonic media. Furthermore, swelling of rat hepatocytes with hypotonic media mimics the System N-stimulatory effects of asparagine. Among the Na(+)-dependent amino acid transport systems present in rat hepatocytes, System N is stimulated preferentially by amino acid-containing or hypotonic media. Collectively, these results demonstrate that cell swelling is a prerequisite for the amino acid-dependent activation of the hepatic System N transporter.     

Effects of medium amino acids on ouabain-sensitive 86Rb+ -uptake and membrane-potential dependent [3H]tetraphenylphosphonium accumulation in Friend erythroleukemia cells

The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells. The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only. The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids. Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin. In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of [3H]TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions. On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of [3H]TPP+-accumulation. These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential.     

Influence of amino acids on hybridoma cell viability and antibody secretion

It is generally accepted that the phase of cell decline observed in batch culture of mammalian cells is related to exhaustion of medium nutrients (principally glucose and glutamine) and/or to waste products accumulation. In the present paper, we have studied the influence of glutamine on the proliferation of mouse hybridoma cells. We showed that repeated addition of glutamine prolonged the life span of the culture and significantly increased the secretion of monoclonal antibody. Flow cytometry analysis suggests that this effect of glutamine is related to a delay in cell death rather than to a stimulation of proliferation.Addition of glutamine and glucose failed however to prevent the death of the culture. Determinations of amino acid consumption in glutamine-supplemented samples and experiments carried out with complementary sources of amino acids (e.g. tryptose phosphate) strongly suggest that amino acid supply is a critical factor governing cell growth and productivity.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.