Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Soft X-ray-induced decomposition of amino acids: an XPS, mass spectrometry, and NEXAFS study

Decomposition of five amino acids, alanine, serine, cysteine, aspartic acid, and asparagine, under irradiation with soft X rays (magnesium Kalpha X-ray source) in ultra-high vacuum was studied by means of X-ray photoelectron spectrometry (XPS) and mass spectrometry. A comparative analysis of changes in XPS line shapes, stoichiometry and residual gas composition indicates that the molecules decompose by several pathways. Dehydration, decarboxylation, decarbonylation, deamination and desulfurization of pristine molecules accompanied by desorption of H2, H2O, CO2, NH3 and H2S are observed with rates depending on the specific amino acid. NEXAFS spectra of cysteine at the carbon, oxygen and nitrogen K-shell and sulfur L2,3 edges complement the XPS and mass spectrometry data and show that the exposure of the sample to an intense soft X-ray synchrotron beam results in the formation of C-C and C-N double and triple bonds. Qualitatively, the amino acids studied can be arranged in the following ascending order of radiation stability: serine相似文献   

Effects of cysteine,asparagine, or glutamine limitations in Chinese hamster ovary cell batch and fed-batch cultures

Amino acid availability is a key factor that can be controlled to optimize the productivity of fed-batch cultures. To study amino acid limitation effects, a serum-free chemically defined basal medium was formulated to exclude the amino acids that became depleted in batch culture. The effect of limiting glutamine, asparagine, and cysteine on the cell growth, metabolism, antibody productivity, and product glycosylation was investigated in three Chinese hamster ovary (CHO) cell lines (CHO-DXB11, CHO-K1SV, and CHO-S). Cysteine limitation was detrimental to both cell proliferation and productivity for all three CHO cell lines. Glutamine limitation reduced growth but not cell specific productivity, whereas asparagine limitation had no significant effect on either growth or cell specific productivity. Neither glutamine nor asparagine limitation significantly affected antibody glycosylation. Replenishing the CHO-DXB11 culture with cysteine after 1 day of cysteine limitation allowed the cells to partially recover their growth and productivity. This recovery was not observed after 2 days of cysteine limitation. Based on these findings, a fed-batch protocol was developed using single or mixed amino acid supplementation. Although cell density and antibody concentration were lower compared to a commercial feed, the feeds based on cysteine supplementation yielded comparable cell specific productivity. Overall, this study showed that different amino acid limitations have varied effects on the performance of CHO cell cultures and that maintaining cysteine availability is a critical process parameter for the three cell lines investigated.     

EPR spectroscopy and theoretical study of gamma-irradiated asparagine and aspartic acid in solid state

Aspartic acid (Asp) and asparagine (Asn) are vulnerable amino acids. One-electron addition or withdrawal reactions initiate many deleterious processes involving these amino acids. To study these redox processes we have irradiated by gamma-rays asparagine or aspartic acid in the solid state. The nature of the resulting free radicals was determined by electron paramagnetic resonance (EPR) and by calculations using DFT methods in various environments. Reactions initiated by electron transfer are different for both amino acids: Asn anion loses hydrogen atom whereas the cation undergoes decarboxylation. Conversely, Asp cation loses hydrogen atom from amine group, which triggers decarboxylation.     

Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.     

Determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides by gas–liquid chromatography

A new procedure for the qualitative and quantitative determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides based on g.l.c. has been developed. Under the conditions of esterification and trifluoroacetylation N-trifluoroacetylaspartic acid mono-n-butyl ester was formed from asparagine and N-trifluoroacetylglutamic acid mono-n-butyl ester from both glutamine and pyrrolidonecarboxylic acid. To distinguish between the latter two compounds, the esterification was carried out at room temperature yielding 30% of esterified pyrrolidonecarboxylic acid but less than 1% of esterified glutamine. In extending the g.l.c. of amino acids, the previously unknown positions in the g.l.c. elution pattern of the following amino acids could also be reproducibly determined: carboxymethylcysteine, homoserine, hydroxylysine and in-methyl-lysine. Further, certain glycopeptides were investigated and the artifacts due to their carbohydrate moieties were determined.     

Amino Acid Pools in Herbaceous Plants at the Wintering Stage and at the Beginning of Growth

Free amino acids in 40 herbaceous perennial plants were analyzedunder natural conditions. From the major amino acid contentat the wintering stage, the pools were separated into the followingfive types: 1) a group which accumulated arginine (20 plantsout of 40); 2) a group which accumulated arginine and proline(9 plants); 3) a group which accumulated glutamate and glutamine(3 plants); 4) a group which accumulated asparagine (4 plants);and 5) a group which accumulated proline (4 plants). Changesin the amino acid pools in the plants occurred under snow duringwintering for about five months. Particularly, asparagine wasno longer the major amino acid in the group which had accumulatedit in fall. There was a tendency for the glutamine content toincrease, suggesting that NH₃ is utilized for the synthesisof the amide. Also, the relative concentrations of almost allthe free amino acids increased several-fold, which was indicativeof the occurrence of biosynthetic processes of general aminoacids during wintering. As the mobile fractions of stored nitrogen,the amino acids appeared to contribute to the initial stageof rapid growth in early spring. (Received August 4, 1986; Accepted November 17, 1986)     

Chicken glucagon: sequence and potency in receptor assay

Glucagon is a 29 amino acid peptide that is generally highly conserved. Among mammalian glucagons the only one that has been shown to differ significantly is that of the guinea pig which differs from the others in 5 of the 9 COOH-terminus amino acids. The amino acid content and partial sequencing of chicken glucagon had been reported earlier. This report describes the purification and complete amino acid sequencing of chicken glucagon and demonstrates that it differs from the usual mammalian glucagon by the replacement of asparagine at position 28 with serine. Chicken glucagon is indistinguishable from porcine glucagon in the rat liver receptor assay system.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Characterization of nitrogen relationships between Sorghum bicolor and the root-hemiparasitic angiosperm Striga hermonthica (Del.) Benth. using K15 NO3 as isotopic tracer

The role of the host in the nitrogen nutrition of Striga hermonthica (Del.) Benth. (Scrophulariaceae) parasitic on Sorghum bicolor cv. SH4 Arval has been investigated using (15)N-nitrate as the tracer. It is shown that, when nitrate is absorbed only by the roots of the host plant, a rapid transfer of nitrogen to the parasite can be detected. The xylem sap of S. hermonthica contained approximately equal amounts of nitrate and amino acids, mostly glutamine and asparagine. Infection altered the free amino acid profile of the host tissues, leading notably to a large increase in asparagine and a decrease in glutamine. The haustoria of S. hermonthica, although rich in nitrate, showed a low concentration of free amino acids, particularly lacking in asparagine and glutamine. The roots of S. hermonthica, in contrast, were rich in both asparagine and glutamine while, in the shoots, asparagine constituted 80% of the total FAA pool. Asparagine was also found to be the primary (15)N-enriched amino acid in the shoots of S. hermonthica while, interestingly, it was glutamate that was most strongly enriched in the roots. It is concluded that nitrogen nutrition in S. hermonthica is based on a supply of both nitrate and amino acids from the host. This implies a non-specific transfer in the transpiration stream. Nitrate reduction probably occurs mainly in the leaves of the parasite. Assimilation also occurs in S. hermonthica and excess nitrogen is stored as the non-toxic nitrogen-rich compound, asparagine. This specific trait of nitrogen metabolism of the parasite is discussed in relation to the effect of nitrogen fertilization on reducing infestation.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Characterization by mass spectrometry of poly(3-hydroxyalkanoates) produced by Rhodospirillum rubrum from 3-hydroxyacids.

The sequence distributions of two microbial copolyesters obtained by fermentation of Rhodospirillum rubrum, grown with 3-hydroxyhexanoic or 3-hydroxyheptanoic acids, were determined by analyzing the oligomers prepared by partial pyrolysis or partial methanolysis of these copolyesters using fast atom bombardment mass spectrometry (FAB-MS). Oligomers up to pentamers were identified in the case of partial pyrolysis and up to tetradecamers in the case of partial methanolysis. The comparison between the experimental and calculated peak intensities of FAB mass spectra allows the calculation of compositions and sequence distributions, which in these copolyesters follow Bernoullian statistics, indicating that they are random terpolyesters.     

Regulation of Growth and Gametangial Formation in Riccia gangetica Ahmad by Some Amino Acids

The present work deals with the effect of six amino acids: asparagine,aspartic acid, glutamic acid, glycine, serine, and tryptophan,on growth and gametangial formation in Riccia gangetica. Allthe amino acids tested enhance vegetative growth, and amongthese glutamic acid proves best. The total number of rhizoidsis reduced in response to amino acids. Aspartic acid and glutamicacid favour antheridial production. In contrast, asparagine,serine, and tryptophan enhance archegonial formation, and amongthese asparagine elicits the best response. Glycine proves bestfor antheridial production, and also increases the number ofarchegonia. Key words: Riccia gangetica, Amino acids, Growth, Gametangial formation     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.     

Origin of Foliar Nitrogen and Changes in Free Amino-Acid Composition and Content of Leaves, Stubble, and Roots of Perennial Ryegrass During Re-growth after Defoliation

Nitrogen re-mobilization and changes in free amino acids werestudied as a function of time in leaves, stubble, and rootsduring ryegrass (Lolium perenne L.) re-growth. Experiments with¹⁵N labelling clearly showed that during the first days nearlyall the nitrogen in new leaves came from organic nitrogen re-mobilizedfrom roots and stubble. On the days of defoliation, stubblehad the highest content of free amino acids with 23 mg per gdry weight against 15 mg and 14 mg in leaves and roots, respectively.The major amino acids in leaves were asparagine (23% of totalcontent in free amino acids), aminobutyrate, serine, glutamine,and glutamate (between 7% and 15%) whereas in roots and stubblethe contribution of amides was high, especially asparagine (about50%). Re-growth after cutting was associated with a rapid increaseof the free amino acid content in leaves, with a progressivedecrease in roots while stubble content remained virtually unchanged.In leaves, asparagine increased from the first day of re-growth,while the aspartate level remained unchanged and glutamine increasedstrongly on the first day but decreased steadily during thenext few days of re-growth. Asparagine in stubble and rootschanged in opposite directions: in stubble it tended to increasewhereas in roots it clearly decreased. In contrast, stubbleand roots showed a similar decrease in glutamine. In these twoplant parts, as in leaves, aspartate remained at a low level.Results concerning free amino acids are discussed with referenceto nitrogen re-mobilization from source organs (stubble androots) to the sink organ (regrowing leaves). Key words: Lolium perenne L, re-growth, nitrogen, free amino acids, glutamine, asparagine     

General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

Changes in Total Nitrogen and Free Amino Acids in Stem Cuttings of Mulberry (Morus alba L.)

Changes in total nitrogen and free amino acid contents in stemcuttings of Morus alba have been studied. The fresh and dryweights and total nitrogen amounts of the parent stems of cuttingsdecreased initially after cutting. Their increase follows theformation of main roots in cuttings, suggesting that, like carbohydrates,sugars and starch, stored nitrogenous substances are used forsprouting and rooting of cuttings. Amino acids found in stems,roots and shoots are those common in other higher plants withthe exception of pipecolic acid and 5-hydroxypipecolic acid.Significant changes in the levels of asparagine, proline, arginine,-aminobutyric acid and alanine in roots, bark and wood of parentstems were observed during cutting growth, whereas those ofother amino acids remained comparatively constant; the mostpredominant amino acid in the starting materials was proline.while that in the cuttings during growth was asparagine. Theresults suggest that, among free amino acids, asparagine, prolineand arginine play the major part in storage of nitrogen in mulberry.The importance of glut-amine and asparagine in nitrogen metabolismin mulberry has been discussed.     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.