Strychnine-Sensitive, Glycine-Induced Release of [3H]Norepinephrine from Rat Hippocampal Slices

The amino acid glycine is the primary inhibitory neurotransmitter of the mammalian spinal cord. Glycine has also been shown to facilitate the excitatory actions of glutamate at the N-methyl-D-aspartic acid receptor subtype. In this article, glycine is shown to increase the Ca2(+)-dependent release of [3H]norepinephrine from preloaded slices of the rat hippocampus. This effect was inhibited noncompetitively by nanomolar concentrations of strychnine, which differentiates it from the glycine site associated with the N-methyl-D-aspartate receptor. Glycine also released [3H]acetylcholine, but was without effect on the efflux of [3H]serotonin or gamma-[3H]aminobutyric acid from the same tissue preparation. The release of [3H]norepinephrine was reversibly blocked by tetrodotoxin, indicating the effect is not initiated at the noradrenergic terminals, but requires propagation of an action potential. The results suggest that a glycine site that is pharmacologically similar to that found in the spinal cord exists in the rat hippocampus. We suggest that this site may participate in modulating the release of specific neurotransmitters in the brain.     

Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.     

Relationship Between Plasma and Brain Large Neutral Amino Acids in Rats Fed Diets with Different Compositions at Different Times of the Day

Large neutral amino acids (LNAAs) compete with each other for carrier-mediated transport through the blood-brain barrier into the brain. The relative plasma concentration, expressed as the ratio of each LNAA to the sum of LNAAs, is considered the main regulator of brain LNAA concentrations. In order to investigate the consistency of this assumption throughout a 24-h period, we have compared the relationship of plasma LNAAs to brain LNAAs among groups of rats fed diets containing various amounts of protein (in order to obtain a wide range of plasma LNAA levels) at two different phases of the light/dark cycle (0900 and 2100 hours). The relationship between plasma and brain LNAAs was found to be dependent on both diet and the time of day. Similar plasma amino acid concentrations in the morning and in the evening contrasted with different brain concentrations. Furthermore, previous findings that brain LNAA concentrations are influenced by plasma amino acid concentrations were confirmed.     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spinal subarachnoid perfusion in the rat: glycine transport from spinal fluid

Abstract— A method was developed for perfusion of the spinal subarachnoid space in the rat. Bidirectional steady-state fluxes of [¹⁴C]glycine between spinal fluid and plasma were measured. [¹⁴C]glycine clearance from spinal fluid was 5-fold greater than its clearance from plasma. Glycine was transported out of spinal fluid by a saturable process, and the rate of transport was unaffected by the other depressant amino acids, GABA, β-alanine, and taurine. Perfused [¹⁴C]glycine and [³H]GABA distributed in an intracellular compartment in spinal cord. The preparation should be useful for study of the release of these inhibitory amino acids from the intact spinal cord.     

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.     

Diurnal rhythms in rat plasma amino acids

To obtain detailed data on the diurnal rhythm in rat plasma amino acids, groups of rats were killed every two hours during 24 hours and the amino acids in plasma were measured. By using such a short interval between the blood samples, it was possible to reveal differences in rhythmicity between the various amino acids, more detailed than those previously described. Furthermore, it was found that those large neutral amino acids (LNAA) which compete with each other for the carrier mediated transport from plasma into the brain demonstrated different rhythms, whereby also the relation between these competing amino acids varied during the day. This finding might have implications for the transport of the various LNAAs into the brain, and secondarily also for the synthesis of the monoaminergic neurotransmitters in the neurons, for which the LNAAs tyrosine and tryptophan serve as precursors.     

Kinetic Constants for Blood—Brain Barrier Amino Acid Transport in Conscious Rats

The kinetic constants for large neutral amino acid (LNAA) transport across the blood-brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 microliters) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.     

Alterations of Amino Acid Transmitter Systems in Spinal Cords of Chronic Paraplegic Dogs

The high-affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid [( 3H]GABA) and the Na+-independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosacral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity uptake and for preparing synaptic plasma membranes for Na+-independent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie-Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high-affinity uptakes of 1 microM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60-70% higher for the spastic group than for the control animals. For both amino acids, Eadie-Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+-independent binding of 5 nM [3H]glutamate was 40-85% higher and the binding of 10 nM [3H]GABA was 40-60% lower for the paraplegic groups relative to the values for the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Measurement of 3,4-dihydroxyphenylacetic acid and 3-methoxytyramine specific activity rat striatum.

A method is described for the simultaneous determination in rat striatum of the specific activities of tyrosine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 3-methoxytyramine (3-MT) after administration of [³H]tyrosine. [³H]Tyrosine was given intraventricularly to nonanesthetized rats, and the animals were killed by exposure to microwave radiations. Combined chromatographic elutions on Dowex 50W-X4 columns and alumina or solvent extractions were devised to separate the compounds. Fluorimetric, mass-fragmentographic, and radiometric techniques were used for their detection. Recovery was 94% for tyrosine, 72% for dopamine, 63% for DOPAC, and 50% for 3-MT. Concentrations of the labeled compounds in rat striatum 15 min after the [³H]tyrosine injection were at least five to eight times higher than background. Identity of the final fractions containing 3-MT and DOPAC tissue extracts was verified by thin-layer chromatography. α-Methyltyrosine pretreatment of rats markedly reduced the formation of labeled dopamine. DOPAC, and 3-MT from [³H]tyrosine.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

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.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

An Excitant Amino Acid Projection from the Medial Prefrontal Cortex to the Anterior Part of Nucleus Accumbens in the Rat

High-affinity uptake of neurotransmitter substrates in synaptosome-containing homogenates and tissue concentrations of amino acids were examined in subcortical areas 5-6 days after bilateral N-methyl-D-aspartate lesions confined to rat medial prefrontal cortex. D-[3H]Aspartate (32% of control) and [3H] gamma-aminobutyric acid ( [3H]GABA) (60% of control) uptakes were significantly reduced in medial prefrontal cortex, whereas [3H]choline (110% of control) uptake was unchanged, suggesting the production of axon-sparing lesions. The uptake of D-[3H]aspartate (76% of control), but not of [3H]GABA or [3H]choline, was significantly reduced in nucleus accumbens, with no concomitant reduction in amino acid concentrations. When examined in serial coronal sections, reduced D-[3H]aspartate uptake was confined to the most anterior 500 micron of nucleus accumbens (67% of contralateral sample). No significant reductions of uptake or amino acid concentrations were observed in caudate putamen or ventral tegmental area. These results suggest a role for glutamate or aspartate as neurotransmitters in projections from medial prefrontal cortex to anterior nucleus accumbens. Medial prefrontal cortex may represent the major excitatory cortical input to the nucleus accumbens.     

Large neutral amino acids: dietary effects on brain neurochemistry and function

The ingestion of large neutral amino acids (LNAA), notably tryptophan, tyrosine and the branched-chain amino acids (BCAA), modifies tryptophan and tyrosine uptake into brain and their conversion to serotonin and catecholamines, respectively. The particular effect reflects the competitive nature of the transporter for LNAA at the blood–brain barrier. For example, raising blood tryptophan or tyrosine levels raises their uptake into brain, while raising blood BCAA levels lowers tryptophan and tyrosine uptake; serotonin and catecholamine synthesis in brain parallel the tryptophan and tyrosine changes. By changing blood LNAA levels, the ingestion of particular proteins causes surprisingly large variations in brain tryptophan uptake and serotonin synthesis, with minimal effects on tyrosine uptake and catecholamine synthesis. Such variations elicit predictable effects on mood, cognition and hormone secretion (prolactin, cortisol). The ingestion of mixtures of LNAA, particularly BCAA, lowers brain tryptophan uptake and serotonin synthesis. Though argued to improve physical performance by reducing serotonin function, such effects are generally considered modest at best. However, BCAA ingestion also lowers tyrosine uptake, and dopamine synthesis in brain. Increasing dopamine function in brain improves performance, suggesting that BCAA may fail to increase performance because dopamine is reduced. Conceivably, BCAA administered with tyrosine could prevent the decline in dopamine, while still eliciting a drop in serotonin. Such an LNAA mixture might thus prove an effective enhancer of physical performance. The thoughtful development and application of dietary proteins and LNAA mixtures may thus produce treatments with predictable and useful functional effects.     

Acute tryptophan and serotonin depletion using an optimized tryptophan-free protein-carbohydrate mixture in the adult rat

In contrast to humans, a tryptophan (TRP)-free amino acid (AA) mixture only leads to moderate depletion in plasma TRP levels in adult rats. In this study we evaluated the effects of an acute administration of a TRP-free protein-carbohydrate nutritional mixture in adult male Wistar rats. Plasma amino acid levels were examined at 2 and 4h starting after the first administration. Furthermore, the concentrations of amino acid, serotonin (5-HT), dopamine (DA) and their metabolite (5-hydroxyindolacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC), respectively) were measured within the striatum, hippocampus and cortex. In the TRP depleted animals, the TRP/sigmaLNAA ratio (LNAA: large neutral amino acids) was substantial decreased at 2 and 4h after the first administration of the oral administration (by 71 and 78%, respectively). Four hours after treatment central TRP and 5-HT concentrations were decreased by 50%. Both peripheral and central TRP levels returned to basal values in the group treated with the nutritional mixture supplemented with TRP. Surprisingly, tyrosine levels were also reduced after oral administration of the protein-carbohydrate mixture without affecting central DA concentrations. In conclusion, the TRP-free protein-carbohydrate nutritional mixture appears to be an efficient tool to substantially reduce plasma and central TRP levels in adult rat.     

Demonstration of an Autoreceptor Modulating the Release of [3H]5-Hydroxytryptamine from a Synaptosomal-Rich Spinal Cord Tissue Preparation

A superfusion system employed to measure the K+-stimulated release of [3H]5-hydroxytryptamine [(3H]5-HT, [3H]serotonin) from a synaptosomal-rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5-HT release. Spinal 5-HT release is apparently modulated by an autoreceptor. Exogenous 5-HT depressed, in a concentration-dependent manner, the K+-stimulated release of [3H]5-HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration-dependent decrease in [3H]5-HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5-HT. The 5-HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5-HT at the spinal 5-HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5-HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5-HT and [3H]5-hydroxyindoleacetic acid ( [3H]5-HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high-affinity [3H]5-HT binding in the spinal cord (designated 5-HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5-HT1B subtype, antagonized the actions of exogenous 5-HT on K+-stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5-HT1A sites for the drug, was ineffective in altering the ability of exogenous 5-HT to modulate K+-stimulated [3H]5-HT release. These results suggest that 5-HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.     

Effect of acute administration of large neutral and other amino acids on urinary excretion of catecholamines

We examined the specificity of tyrosine's ability to increase catecholamine excretion by rats. Tyrosine alone among amino acids tested caused major increases in tissue and serum tyrosine, as well as urinary catecholamine levels. Large neutral amino acids (tryptophan, valine or isoleucine) and representatives of other classes of amino acids (glutamate, alanine, lysine or arginine) were unable to mimic tyrosine's action.     

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.