Axonal microtubules necessary for generation of sodium current in squid giant axons: II. Effect of colchicine upon asymmetrical displacement current

Effect of internal colchicine on asymmetrical displacement currents was studied by internally perfusing squid giant axons with a solution containing colchicine. It was found that (1) asymmetrical displacement currents were composed of two parts; colchicine-sensitive and colchicine-resistant; that (2) the colchicine-sensitive part had a definite rising phase while the colchicine-resistant one showed an instantaneous jump, followed by exponential decay; and that (3) the colchicine-sensitive part related to normal Na channels.     

Dominance of colchicine resistance in hybrid CHO cells

Intraspecific hybrids of colchicine-sensitive with colchicine-resistant (CHR) Chinese hamster ovary cells were constructed, using six different colchicine-resistant clones from two independent series. In each instance, colchicine resistance was expressed in an incompletely dominant manner. Some hybrid clones were examined further for the expression of the pleiotropic CHR phenotype and for the cell surface P glycoprotein. These features of the colchicine-resistant phenotype were also expressed coordinately.     

Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.     

THE METABOLISM OF TISSUE CULTURES : I. PRELIMINARY STUDIES ON CHICK EMBRYO

1. The metabolism of chick embryo tissues has been followed by analysis of the culture media after various periods of incubation in roller bottles. 2. The initial rate of glucose utilization is increased by increasing glucose in the medium from 100 to 500 mg. per cent. Total glucose used can be increased in the same way or by daily addition of small amounts. Glucose is used in greatest amount when the medium containing 100 mg. per cent is replaced daily. 3. Although glucose consumption appears necessary for survival of cultures it may be used at a rate far in excess of that required for life and maximal growth. Complete blocking of mitosis by colchicine does not alter the rate of glucose utilization. 4. Proteolytic activity of the cultures is shown by an increase in the amino nitrogen of the peptone medium after incubation with tissue. 5. Utilization of nitrogen from an amino acid medium is shown by a decrease in the amino nitrogen of this medium. Cells obtaining their nitrogen from amino acids proliferate as rapidly as those grown in a medium identical except for the substitution of peptone, but the cell type is markedly different, in that embryo muscle forms cells resembling regenerating adult muscle. 6. Lactic acid was formed in both the presence and absence of glucose. Its formation increased with increased glucose utilization. There is some evidence that lactate may be utilized, and that it favors growth in the absence of glucose. 7. Added pyruvate was rapidly metabolized by the tissues. It, too, favors growth slightly in the absence of glucose.     

Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.     

Low Contents of Carbon and Nitrogen in Highly Abundant Proteins: Evidence of Selection for the Economy of Atomic Composition

Proteins that assimilate particular elements were found to avoid using amino acids containing the element, which indicates that the metabolic constraints of amino acids may influence the evolution of proteins. We suspected that low contents of carbon, nitrogen, and sulfur may also be selected for economy in highly abundant proteins that consume large amounts of the resources of cells. By analyzing recently available proteomic data in Escherichia coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, we found that at least the carbon and nitrogen contents in amino acid side chains are negatively correlated with protein abundance. An amino acid with a high number of carbon atoms in its side chain generally requires relatively more energy for its synthesis. Thus, it may be selected against in highly abundant proteins either because of economy in building blocks or because of economy in energy. Previous studies showed that highly abundant proteins preferentially use cheap (in terms of energy) amino acids. We found that the carbon content is still negatively correlated with protein abundance after controlling for the energetic cost of the amino acids. However, the negative correlation between protein abundance and energetic cost disappeared after controlling for carbon content. Building blocks seem to be more restricted than energy. It seems that the amino acid sequences of highly abundant proteins have to compromise between optimization for their biological functions and reducing the consumption of limiting resources. By contrast, the amino acid sequences of weakly expressed proteins are more likely to be optimized for their biological functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

The fenpropimorph resistance gene FEN2 from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenate symporter.

The product of the FEN2 gene of Saccharomyces cerevisiae has previously been described as a protein conferring sensitivity to the antifungal agent fenpropimorph. Fen2p was postulated to act as a common regulator of carbon and nitrogen catabolite repression and of amino acid and ergosterol biosynthesis. In this paper, we present experimental evidence characterizing Fen2p as a plasma membrane-localized transporter for the vitamin pantothenate. The high affinity transport system (Km = 3.5 microM) is sensitive to uncouplers, suggesting a H+-pantothenate cotransport. Pantothenate transport rates in yeast are modulated by extracellular pantothenate, being maximal at low pantothenate concentrations. It is demonstrated that beta-alanine can suppress the growth defect of FEN2 wild-type and fen2 mutant cells on pantothenate-free medium. Evidence is presented that beta-alanine is transported by the general amino acid permease Gap1p. The relation among pantothenate transport, nitrogen catabolite repression, and sensitivity to the antifungal agent fenpropimorph is discussed.     

Stimulation of protein synthesis by lysine analogues in lysine-deprived Ehrlich ascites tumour cells

This paper describes experiments in which we have investigated the mechanism by which amino acid starvation regulates the initiation of protein synthesis in mammalian cells. We have examined the ability of a range of lysine analogues to stimulate protein synthesis in lysine-deprived mouse Ehrlich ascites tumour cells in culture. Of those analogues tested, only those which are cleaved to lysine intracellularly are capable of restoring protein synthesis to the level seen in fully fed cells. Lysine which is covalently linked to agarose does not stimulate translation. After 5 min incubation of lysine-deprived cells with the analogue lysine p-nitroanilide, the lysine concentration in cell extracts is restored to that found in extracts from fed cells, and protein synthesis is maximally stimulated within 5–10 min. During this period of time there is no increase in the concentration of lysine in the medium. These data indicate that it is the size of the intracellular rather than the extracellular amino acid pool which regulates the rate of protein synthesis during amino acid deprivation.     

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.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Changes in Free Amino Acid Production and Intracellular Amino Acid Pools of Bacillus licheniformis as a Function of Culture Age and Growth Media

Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed.     

DNA-protein cross-links as a possible reason for genomic damage during its protonation]

The change in survival of bacteriophages with DNA of different GC-contents after their incubation in media of different acidities with subsequent neutralization was studied. It was shown that the higher the GC-content, the more sensitive is the phage to the action of H(+)-ions. Evidence is presented that the acidic inactivation of virions is not connected with the helix-coil transition of the intraphage DNA due to its protonation. The extractability of DNA from phages subjected to different concentrations of H(+)-ions with subsequent neutralization of the medium to pH 8 was determined. The changes in: transfection ability, UV-spectra, the quantity of the residual proteins, and the contents of glutamic and lysine amino acid residues in these proteins were investigated. The effect of glutamic acid on the parameters of DNA melting curves was followed for different pH values. Proceeding from the data obtained, we concluded that acidification of the medium from neutral tp pH congruent to 4 leads to formation of non-covalent DNA-protein cross-links due to interaction of the GC base pairs of DNA with glutamic and aspartic amino acid residues, whereas acidification of the medium to pH less than 4 with subsequent neutralization to pH 8 results in the formation of covalent DNA-protein cross-links of Schiff base type. The influence of non-covalent DNA-protein cross-links on the properties of DNA and their regulatory role in genome functioning are discussed.     

Amplification of portions of the genome in mammalian somatic cells resistant to colchicine. VI. Restriction analysis of the amplified DNA sequences

Fragments specific for the amplified regions in DNA of Djungarian hamster colchicine-resistant cells were studied after restriction endonuclease digestion. We used three different methods of detection of these fragments: a) comparison of the wild type and resistant cell DNA electroforegramms stained by ethidium bromide; b) blotting of DNA from sensitive and resistant variants onto nitrocellulose filters and their hybridization with nick-translated DNA from resistant cells, in the presence of the excess of unlabelled DNA from the wild type cells (competitive hybridization); c) investigation of autonomously replicating DNA from sensitive and colchicine-resistant sublines. The highest resolution was found using the third method. However, the competitive hybridization is evidently a more universal approach to restriction analysis of DNA amplified sequences, because it gives quite high resolution and may be used for studying both autonomously and non-autonomously replicating sequences.     

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.     

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

Antizyme-dependent and -independent mechanisms are responsible for increased spermidine transport in amino acid-restricted human cancer cells

Amino acid deprivation can inhibit tumour cell proliferation. Since polyamines are required for cell growth, we hypothesised that their regulatory pathways can respond to amino acid restriction. We report here that exposure of human colon adenocarcinoma Caco-2 cells to a medium restricted for a single amino acid, but not for D-glucose, activates spermidine transport. The increase was rapid and seemed transient with a maximum 4-6 hr after amino acid removal. Kinetics showed that the maximal velocity of transport was solely increased in L-methionine- or L-leucine-deprived cells, indicating increased number of transporters. The intracellular level of complex of ornithine decarboxylase (ODC) with antizyme, a negative regulator of polyamine transport, was decreased by 16-29% in amino acid-deprived cells. However, exposure to limited amounts of amino acid increased transport without altering the ODC-antizyme complex level. We propose that antizyme-independent mechanisms, sensitive to the amino acid concentration, also participate to the control of spermidine transport.