Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

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.     

TRANSPORT OF TYROSINE, PHENYLALANINE, TRYPTOPHAN AND GLYCINE IN NEUROBLASTOMA CLONES

Amino acid transport was studied in three neuroblastoma clones, N-TD6, which synthesizes norepinephrine, N-T16, which synthesizes small amounts of serotonin, and N-S20Y, which synthesizes acetylcholine. All three clones exhibited high-affinity saturable transport systems for tyrosine, phenylalanine, tryptophan and glycine as well as systems unsaturated at amino acid concentrations of 1 mM in the external medium. Tyrosine, phenylalanine and tryptophan enter all three clones by rapidly exchanging transport systems which appear to be relatively insensitive to lowered external [Na⁺] or to the presence of 2,4-dinitrophenol (DNP). Glycine uptake was slower and was much more sensitive to lowered external [Na⁺] and to the presence of DNP in the medium. Glycine transport in N-T16 cells was decreased more markedly at low temperature than was transport of the three aromatic amino acids. K_m and V_max values found for saturable transport of tyrosine, phenylalanine and tryptophan were sufficiently low to suggest that, if similar amino acid transport systems exist in neuronal membranes, and if amino acid levels in brain extracellular fluid are similar to levels in plasma, such systems may serve, in conjunction with transport systems in cerebral capillaries, to limit the entry of amino acids into brain cells when blood amino levels are near the normal physiological range.     

Amino Acid Composition of Elm Seeds

In the biosynthetic pathway of aromatic amino acids of Brevibacterium flavum, ratios of each biosynthetic flow at the chorismate branch point were calculated from the reaction velocities of anthranilate synthetase for tryptophan and chorismate mutase for phenylalanine and tyrosine at steady state concentrations of chorismate. When these aromatic amino acids were absent, the ratio was 61, showing an extremely preferential synthesis of tryptophan. The presence of tryptophan at 0.01 mM decreased the ratio to 0.07, showing a diversion of the preferential synthesis to phenylalanine and tyrosine. Complete recovery by glutamate of the ability to synthesize the Millon-positive substance in dialyzed cell extracts confirmed that tyrosine was synthesized via pretyrosine in this organism. Partially purified prephenate aminotransferase, the first enzyme in the tyrosine-specific branch, had a pH optimum of 8.0 and Km’s of 0.45 and 22 mM for prephenate and glutamate, respectively, and its activity was increased 15-fold by pyridoxal-5-phosphate. Neither its activity nor its synthesis was affected at all by the presence of the end product tyrosine or other aromatic amino acids. The ratio of each biosynthetic flow for tyrosine and phenylalanine at the prephenate branch point was calculated from the kinetic equations of prephenate aminotransferase and prephenate dehydratase, the first enzyme in the phenylalanine-specific branch. It showed that tyrosine was synthesized in preference to phenylalanine when phenylalanine and tyrosine were absent. Furthermore, this preferential synthesis was diverted to a balanced synthesis of phenylalanine and tyrosine through activation of prephenate dehydratase by the tyrosine thus synthesized. The feedback inhibition of prephenate dehydratase by phenylalanine was proposed to play a role in maintaining a balanced synthesis when supply of prephenate was decreased by feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP*) synthetase, the common key enzyme. Overproduction of the end products in various regulatory mutants was also explained by these results.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na⁺-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na⁺. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

Tryptophan Metabolism in Ochromonas malhamensis

Tryptophan enhanced the growth of Ochromonas malhamensis at concentrations up to 0.4 mg/ml; higher concentrations inhibited, the growth inhibition being reversible by tyrosine and adenine. The presence of a tryptophan synthetase system in vitro was demonstrated. Tyrosine and phenylalanine stimulated the activity of this enzyme. The uptake of exogenous tryptophan was accompanied by an increase in the free tryptophan pool which in turn suppressed the tryptophan synthetase system, thus pointing to a controlled mechanism. Incorporation of tryptophan in the growth medium enhanced the biosynthesis of folate-active compounds. An elucidation of the mode of action of tryptophan is attempted on the basis of known metabolic pathways.     

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Nutritional Requirements of Plasmodium falciparum in Culture. I. Exogenously Supplied Dialyzable Components Necessary for Continuous Growth

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na₂HPO₄, Ca(NO₃)₂, MgSO₄, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.     

Utilization of tyrosine and tryptophan for protein synthesis by undernourished developing rat brain

Incorporation of tracer doses of radiolabeled tryptophan and tyrosine into brain proteins was investigated in rats malnourished during gestation and lactation. Age and time dependent increases in the radioactivity was observed in the whole homogenate and in the TCA insoluble fraction. Protein malnourished rats showed increased incorporation of tryptophan and tyrosine. However the diet restricted (Pair-fed) animals showed increased incorporation of tyrosine only. The increased incorporation may probably be due to changes in the pool size of the amino acids and effective recycling of the amino acids. The enhanced utilization in protein synthesis may also probably offer a mechanism for conservation of these amino acids.     

Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera,curculionidae) to a diet supplemented with aromatic amino acids

Developmental times of symbiotic and aposymbiotic strains of the rice weevil Sitophilus oryzae were compared, in response to changes in concentration of phenylalanine or tyrosine in whole wheat flour pellets. Aposymbiotic insects were shown to require more aromatic amino acids than symbiotic insects, since a very low supply (0.1%) resulted in faster growth (11%). Incorporation results of [³H]-tyrosine during the larval and pupal stages indicated that total tyrosine intake was lower in aposymbiotic insects, but the incorporation into the cuticle of both strains did not significantly differ. It is suggested that the slower growth rate of weevils without symbiotes is due, in part, to a less efficient utilization of exogenous tyrosine (in the food) and to a lack of endogenous tyrosine (supplied by the symbiotes).     

THE EFFECT OF l-DOPA AND AN INHIBITOR OF PERIPHERAL DECARBOXYLATION ON GLUCOSE METABOLISM IN BRAIN1

Abstract– The effect of the administration of l -DOPA plus an inhibitor of peripheral l -aromatic amino acid decarboxylase (aromatic-l -amino-acid carboxy-lyase; EC 4.1.1.28) on the metabolism of glucose in brain was studied by administering [U-^I4C]glucose (20μCi) to three groups of rats: (1) rats that had been injected with l -DOPA (200mg/kg) 28min earlier; (2) rats that had been similarly injected with l -DOPA and also with N-(d,l -seryl)-N′-(2,3,4-trihydroxybenzyl)hydrazine (50 mg/kg), an inhibitor of l -aromatic amino acid decarboxylase, 30min before the l -DOPA; and (3) appropriate controls. The flux of ¹⁴C from glucose in plasma to those amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates was reduced by treatment with l -DOPA and reduced further by treatment with l -DOPA and the decarboxylase inhibitor. Concentrations of glucose in brain and in plasma were increased after treatment with l -DOPA; these increases were attenuated if the inhibitor was given before the l -DOPA. After treatment with l -DOPA, there were decreases in the concentration of aspartate, tryptophan, and tyrosine in brain. After the administration of l -DOPA and the decarboxylase inhibitor, the concentrations in brain of alanine, glutamate, tyrosine, and phenylalanine were greater, and the concentrations of aspartate, leucine, lysine, histidine, arginine, and tryptophan were less than in control rats.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

Nitrogen assimilation by a Citrus tree

Primary assimilation of ¹⁵N-ammonium or ¹⁵N-nitrate by excised leaves of satsuma mandarin (Citrus unshiu Markovitch) was examined under light and dark conditions. Under both conditions both types of nitrogen were most markedly incorporated into glutamine-amide nitrogen in the primary step of the assimilation, and into proline in the later steps. Incorporation of ammonium or nitrate into amino acids was more active in the light than in the dark, although the stimulating effect of light on the incorporation was relatively small in both cases.