Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

Na+-dependent amino acid transport is a major factor determining the rate of (Na+,K+)-ATPase mediated cation transport in intact HeLa cells

Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane.     

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

The role of free amino acids in osmoregulation of cricket blood (Acheta domesticus)

The blood volume increased during normal feeding, and did not decrease during fasting at the end of the stadium. The unexpectedly high blood volume of starved crickets might be an adaptation to increase chances for moulting via stretch receptor stimulation.The amount of blood amino acids was not changed by feeding, but increased with fasting or starvation. Thus amino acid levels in the blood were not directly related to amino acid input from the gut.The blood protein concentration did not change during starvation, but the amino acid concentration was 33% higher in starved crickets that drank water as opposed to those given saline to drink. Thus amino acid levels in the blood were not related directly to blood protein concentration.The blood amino acid concentration was 19–22 mM/l in response to salt intake by feeding crickets or starved crickets drinking saline. The concentration was 32–38 mM/l when the crickets were fasting prior to and after ecdysis or when starved with water to drink during the time when they would normally be feeding. The increase of amino acids during fasting was due to a proportional increase in all amino acids augmented by a 3 × increase in tyrosine. The increase during salt depleting starvation was due to a doubling of the two predominant amino acids proline and glycine. Proline and glycine were not increased in starved crickets drinking saline, thus starvation was not the reason for the increase. This is the first instance where specific amino acids have been implicated in osmoregulation.     

Patterns of short-term amino acid accumulation and loss in the root-zone of liquid-cultured forage rape (Brassica napus L.)

Amino acid release from roots of sterile and non-sterile, solution-grown, 7-, 21- and 60-days-old forage rape plants (Brassica napus L.), was measured over periods of up to 6 hours. With sterile plants, release of amino acids into a fixed volume of collection medium (6, 12, 70 mL) was concentration-limited, giving rise to similar convex accumulation profiles for individual acids. In contrast, amino acid accumulation in continuously circulating collection medium was not concentration limited, giving a linear accumulation pattern. The compositions of accumulating amino acids, which were similar to those measured in root extracts, did not change significantly. However, the proportions of ALA, GABA, GLU and ILE in both root extracts and root-derived amino acids increased as plants aged. Older plants released more amino acids per plant, while younger plants released more amino acids g^-1 root DW. Using non-sterile plants, the patterns of change in amino acid concentration and composition in the collection medium were completely different from those determined with sterile plants. In general, with 7-days-old plants, and 60-days-old plants that had recently become non-sterile, an initial rise in the concentration of all acids was followed by a fall to low levels. The loss of amino acids was apparently due to microbial consumption. Individual amino acids attained maximum concentration at different times during the collection process. This is attributed mainly to concentration-dependent differential assimilation of amino acids, since those with the highest initial concentrations, the major components of the mixtures released from roots, declined the earliest. When calculated rates of amino acid release from roots (R_r) and microbial consumption of amino acids (R_c) were compared (for 7-days-old plants), the highest ratios of R_c/R_r were found for ASN, ARG, GLU, GLN, and LYS. This suggests a degree of selectivity for glutamate and nitrogen-rich acids on the part of the consuming micro-organisms. With 21-days old plants and 60-days old plants grown entirely under non-sterile conditions, fluctuations in amino acid concentration were similar for all acids.     

Optimization of the culture medium composition for the antibody response of mouse spleen cells

Summary The Mishell-Dutton culture system for in vitro primary antibody response of mouse spleen cells was used to optimize the amino acid composition of RPMI 1640 media. Each of the 20 amino acids was tested over a broad range of concentrations always leaving the remaining 19 amino acids unaltered (i.e. at the formula recommended concentration). In several instances, higher plaque-forming cell responses were obtained with an amino acid concentration that was either higher or lower than that recommended: (a) the optimum concentration for valine, glutamine, and lysine lies considerably above the recommended one, (b) the optimum concentration for leucine as well as for several other amino acids lies below the recommended concentration, and (c) the optimum concentration for arginine corresponds exactly to the recommended concentration. The second round of optimization, i.e. combining of two conditions that individually yielded an improved response often caused a decrease of response. The possibility is discussed that for an optimal response a ratio of two or several amino acids rather than the absolute concentration of any one amino acid is of importance. The Basel Institute for Immunology was founded and is supported by F. Hoffman-La Roche & Co., Ltd.     

Peptide analogs of the mud crab pumping pheromone: structure-function studies

In the mud crab, Rhithropanopeus harrisii (Gould), larval releaseis highly synchronous and is controlled by peptide pheromonesreleased from the hatching eggs. The pheromones and syntheticpeptide analogs, containing neutral amino acids at the arnino-terminusand a basic amino acid at the carboxy-terminus, evoke larvalrelease or pumping behavior in the female. Here we describestructure–function relationships between behavioral responsesand peptide analogs of the pumping pheromone. Compounds testedincluded 11 peptides containing 1–7 neutral amino acidresidues and a carboxy-terminal arginine residue, and modifiedarginine compounds. Complex feeding-stimulant mixtures and groupsof peptides were tested to provide information about the numberof different types of receptors. Pumping behavior was describedin terms of response threshold, maximum percentage responseand effective concentration range. All carboxy-terminal argininepeptides tested evoked pumping responses. Response thresholdsranged from 10^-15 M for the most potent peptide Ile-Gly-Argto 10^-8 M for the least potent Tyr-Arg. Effective concentrationranges for the peptides varied from one to four orders of magnitudeand the responses normally declined with further increase inconcentration. Maximum percentage response was largely independentof response threshold and ranged from 30–60%, comparableto assay responses of larval hatch water. Responses within sizegroups of peptides were more similar than between groups. Studieswith modified arginines showed that hydrophobic structures similarin size and shape to small peptides evoked responses. Stericallyhindered hydrophobic moieties were ineffective. Tests with mixturesof active compounds support the hypothesis that there is onetype of pumping peptide receptor.     

Reciprocal inhibition of umbilical uptake within groups of amino acids

Eight pregnant sheep were infused with two amino acid mixtures of different composition: essential amino acids only and the essentials plus some of the nonessentials. Uterine and umbilical uptakes of amino acids were measured before and during infusion. For most of the amino acids, the infusion increased both maternal plasma concentration and umbilical uptake. However, depending on the infusate composition, the increase in maternal concentration of some amino acids was associated with no change or a significant reduction in umbilical uptake. Data were pooled from this and other, similar studies to test the hypothesis that umbilical uptake of several amino acids can be inhibited by coinfused amino acids. The test consisted of fitting the data, by means of multiple regression analysis, to the linear transformation of a saturation kinetics equation in which uptake is assumed to depend on maternal arterial concentrations. The analysis showed significant inhibitory effects within the neutral essential amino acids group and within the lysine-arginine group, with no demonstrable interaction between the two groups. Uterine uptakes did not show clear evidence of saturability and inhibitory interactions, suggesting a large transport capacity and low transporter affinity on the maternal surface of the trophoblast. We conclude that the transport of any given amino acid from placenta to fetus is a function of both its own maternal concentration and the maternal concentration of inhibitory amino acids.     

Altered blood amino acid distribution in genetically obese mice.

The present study was undertaken to determine whether the alteration in amino acid distribution between the plasma and cellular compartment of the blood, previously described in dietary-obese rats, also occurs in genetically obese mice. The blood concentration of individual amino acids and its distribution between plasma and cells of lean and genetically obese mice (ob/ob) have been measured. The results demonstrated that genetically obese mice showed a decrease (55%, P = 0.0489) of free amino acids in the blood cells. Most amino acids were affected and among the most noteworthy characteristics was the observation that the reduction in concentration was more pronounced for the total concentration of the essential amino acids which was reduced by 76% (P = 0.0112) compared to cells of lean mice. These results suggest that an altered amino acid distribution between plasma and blood cells is a consequence of both diet-induced and genetic obesities.     

Larval release in the crab Rhithropanopeus harrisii (Gould): chemical cues from hatching eggs

Decapod crustaceans have rhythmic larval release patterns. Inthe case of Rhithropanopeus harrisii substances associated withhatching eggs induce ovigerous crabs to exhibit stereotypiclarval release behavior involving vigorous abdomen pumping.In this study, the pumping response of ovigerous crabs was usedto investigate the chemistry of the substances (pumping factor)that evoke larval release behavior. Pumping was induced by polarsubstances released into sea water upon egg hatching. Pumpingfactor was concentrated and desalted by adsorption chromatographywith Amberlite XAD-7 resin. Size fractionation by cascade pressuredialysis indicated that most of the active material had a mol.wt. <500 daltons. Biological activity was destroyed by incubationof pumping factor with a non-specific protease. Amino acid analysisof hydrolyzed factor indicated that arginine comprised –50%of the amino acids. Pumping responses were elicited experimentallyby abnormally high concentrations of mixtures of the two majoramino acids (arginine and glycine) in the hydrolysate. Theseresults suggest that pumping factor is a heterogeneous groupof small peptides each containing arginine.     

Patterns of amino acid utilization by aquatic hyphomycetes

Summary The utilization of amino acids in leaf protein and leaf leachate by aquatic hyphomycetes was studied during decomposition in a combined field and laboratory experiment. Leaves were sampled from a stream which exhibited a seasonal variation in free amino acid concentration in surface water, reaching peaks in autumn and winter. In the leaf drift environment the concentration of amino acids was approximately two orders of magnitude higher than in surface water. Protein amino acid content, which was higher in alder leaves than in beech leaves, decreased exponentially and faster in alder leaves, so that protein amino acid content was similar in the two leaf types after 9–10 weeks decomposition. From 55% to 75% of leaf amino acids were used instantaneously by attached fungi, which grew well, especially on alder leaves, regardless of the presence of a grazing amphipod. If nitrogen was a limiting nutrient source for fungi, it appeared to be more advantageous to colonize alder leaves. Four times more fungal species were found on alder leaves than on beech leaves. The changes in concentration of amino acids in leaves and water was described by a set of differential equations. Rate constants for the transfer of amino acids from leaves and water were estimated from experimental data and the preference in fungi for protein-bound and free amino acids evaluated.The amounts of free amino acids in water absorbed by fungi varied between leaf types and leaves at different stages of decay. Experimental data showed a switching behaviour in fungal absorption of dissolved amino acids so that absorption became superproportional at a certain proportion of free amino acids available in the water.     

Effect of exogenous amino acids,glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the -amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.     

Infusion of amino acids decreases the hyperglycaemic effect of per- and postoperative glucose infusion: an experimental study in the rabbit

The effects on blood glucose concentration of 10% glucose with amino acid solution at low (18%) and high (25%) concentration of branched chain amino acids were studied, two days after laparotomy in fasted rabbits. During the whole study period, saline infusion was associated with normoglycaemia. Among the other infusion combinations peroperative infusion of amino acid solution high in branched chain amino acids with 10% glucose was the least hyperglycaemic. An amino acid solution low in branched chain amino acids with 10% glucose had the lowest hyperglycaemic effect 24 hours postoperatively. After 48 hours an infusion of 10% glucose produced the weakest hyperglycaemia. In preoperatively and 24 hours postoperatively 10% glucose was more hyperglycaemic than 10% glucose with the two amino acid solutions. After 24 and 48 hours, infusion of 10% glucose with amino acids high in branched chain amino acids was more hyperglycaemic than 10% glucose with amino acids low in branched chain amino acids.     

AMINO ACID DISTRIBUTION IN THE NERVOUS SYSTEM OF THE CRAB, CARCINUS MAENAS (L.)

The free amino acid levels in the peripheral nerve of the crab, Carcinus maenas, have been measured using various extraction procedures. A comparison of the amino acid levels of the peripheral and central nervous system of the crab has revealed marked differences in the levels of certain amino acids. Aspartate and glutamate were much lower in central nervous tissue than peripheral whilst glycine was much higher. The results are discussed in relation to the extreme steepness of the concentration gradient for amino acids across the neuronal membranes of crustaceans and a comparison is made with other groups. The significance of the differences in amino acid concentration in specific areas of nervous tissue is discussed in relation to the functions of the amino acids and to possible transmitter activity.     

气相色谱-质谱法测定中药沙苑子中的氨基酸

本文利用气相色谱一质谱(GC/MS)联用技术首次对中药沙苑子中的氨基酸进行了定性定量分析。沙苑子经盐酸水解后,对其氨基酸水解液进行羟基脂化和氨基酰化的衍生化反应,利用GC/MS法共测得15种氨基酸,由外标法测定了每种氨基酸的含量。由此得出沙苑子中氨基酸的总质量分数为4.23％。     

Plasma amino acid levels in rainbow trout (Oncorhynchus mykiss)

The time course of plasma amino acid concentrations was studied in adult rainbow trout (300 g mean body weight). After a starvation period of 2 days fish were force-fed either with fish protein concentrate or a mixture of acidic casein and Na-caseinate at a rate of 0.32% CP (N' 6.25) of body weight. Peak levels occurred for feeding fish protein concentrate 6–12 h and for the casein mix 18 h post-feeding. The increase of the essential amino acids was closely correlated to the amino acid profile of the test proteins, whereas the concentration differences of the non-essential amino acids were at no time correlated to the amino acid pattern of fish protein concentrate or even negatively correlated in case of casein. The limiting amino acids in the test proteins were determined by ranking the average concentration increases (decreases) of the individual essential amino acids. Accordingly, arginine and histidine were most deficient in casein; in fish protein concentrate tryptophan seems to be the first limiting amino acid, followed by isoleucine.     

Effect of Gamma Irradiation on the Amino Acid of Potatoes

The free amino acids of potatoes irradiated with the doses of 7,000, 15,000 and 30,000 rad were determined by ion-exchange chromatography.

After 15 days storage following irradiation, it was shown that the concentration of asparatic acid, proline and aliphatic amino acids increased with increasing irradiation doses, while that of basic amino acids and glutamic acid especially decreased. However, after 105 days of storage, the similarity of the free amino acid content of irradiated potatoes to that of non-irradiated and non-stored potatoes was observed.

On the concentration of protein-bound amino acids, there were no significant differences between non-irradiated and 15,000 rad irradiated potatoes.     

Capillary electrophoresis for the determination of major amino acids and sugars in foliage: application to the nitrogen nutrition of Sclerophyllous species

Amino acids and sugars are probably the most commonly measured solutes in plant fluids and tissue extracts. Chromatographic techniques used for the measurement of such solutes require complex derivatization procedures, analysis times are long and separate analyses are required for sugars and amino acids. Two methods were developed for the analysis of underivatized sugars and amino acids by capillary electrophoresis (CE). Separation of a range of sugars and amino acids was achieved in under 30 min, with good reproducibility and linearity. In general, there was close agreement between amino acid analyses by CE and HPLC with post-column derivatization. An alternative, more rapid method was optimized for the common neutral sugars. Separation of a mixture of fructose, glucose, sucrose, and fucose (internal standard) was achieved in less than 5 min. How the source of N applied (nitrate or ammonium) and its concentration (8.0 or 0.5 mM) affects the amino acid and sugar composition of leaves from Banksia grandis Willd. and Hakea prostrata R. Br. was investigated. The amino acid pool of Banksia and Hakea were dominated by seven amino acids (aspartic acid, glutamic acid, asparagine, glutamine, serine, proline, and arginine). Of these, asparagaine and glutamine dominated at low N-supply, whereas at high N-supply the concentration of arginine increased and dominated amino-N. Plants grown with nitrate had a greater concentration of proline relative to plants with ammonium. In Banksia the concentration of amides was greatest and arginine least with a nitrate N-source, whereas in Hakea amides were least and arginine greatest with nitrate N-source. The concentration of sugars was greater in Banksia than Hakea and in both species at greater N-supply.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy

Fulminant hepatic failure was induced in rabbits by intravenous administration of galactosamine hydrochloride. The animals were sacrified after 45 h and the hippocampus analyzed for free amino acids. In addition, free amino acids were measured in plasma and in the extracellular fluid of the hippocampus 20, 30 and 45 h after galactosamine injection. The extracellular fluid compartment was analyzed by slow perfusion of a thin dialysis tube which was implanted in the hippocampus one day prior to galactosamine administration. The amino acid concentration in the extracellular fluid agreed fairly well with that of the cerebrospinal fluid in the control situation. During development of hepatic failure, the plasma concentrations of all amino acids increased. The changes in extracellular amino acids were smaller, except for phosphoethanolamine and glutamate. The concentration ratio intra/extracellular amino acids decreased in the hippocampus for amino acids with a normally high concentration gradient.