Tyrosine Transport Research Articles

Amino-acid transport and protein synthesis in the rabbit lens: absence of cryoprobe effect.

Recently developed organ-culture techniques have been used to investigate the effects of cryoprobe treatment on rabbit lenses. Uptake of 14C-tyrosine into cryoprobe treated and control lenses was followed for 96 h. Lens proteins were separated by gel filtration and incorporation of label measured in the individual crystallins. The cryoprobe treatment had no measurable effect on lens water, Na+, K+ or Ca++ content, tyrosine transport or the incorporation of tyrosine into the crystallins, during the period of the experiment.

Inhibition by L-phenylalanine of tyrosine transport by synaptosomal plasma membrane vesicles: implications in the pathogenesis of phenylketonuria.

The effect of L-phenylalanine on the transport of tyrosine was studied using membrane vesicles from rat brain synaptosomes. Phenylalanine, which is accumulated in phenylketonuria, competitively inhibits tyrosine uptake at concentrations similar to those found in phenylketonuric patients, with a Ki of the same order of the Km for tyrosine. This inhibition could be responsible for the depletion of catecholamines observed in phenylketonuria.

Studies on the transport of tyrosine, leucine, and methionine in cultured B-16 mouse melanoma cells.

1. Linear uptake of tyrosine lasted for 1 min in cultured B-16 mouse melanoma cells preloaded with an amino acid such as tyrosine. 2. The tyrosine uptake increased markedly on preincubating the cells with 0.1 mM methionine, tyrosine, histidine or tryptophan and moderately with 1 mM phenylalanine, valine, isoleucine, or leucine. The effects of the preincubation on leucine uptake were similar to those on tyrosine uptake. 3. The tyrosine uptake was Na-independent under the experimental conditions used (0.05-1.0 mM); Km 75 micrometers and Vmax 15 nmol/min/mg protein. The methionine uptake (0.1-0.5 mM) was also Na-independent (Km 150 micrometers and Vmax 25 nmol/min/mg protein), whereas Na-dependent uptake could contribute at 2 mM methionine. 4. Inhibitions of tyrosine uptake by tryptophan, phenylalanine, leucine, isoleucine, methionine, valine, and histidine were competitive, giving K1 values of 70, 80, 100, 130, 160, 350, and 900 micrometers, respectively. 5. Exchange between intracellular methionine and extracellular tyrosine and vice versa was equimolar. Potencies of amino acids in stimulating tyrosine efflux were in the following order: methionine greater than tyrosine greater than histidine greater than tryptophan greater than phenylalanine greater than leucine greater than isoleucine much greater than valine. 6. The amino acid affinity of the system in the intracellular surface was suggested to be different from that in the extracellular surface. 7. The theophylline-treated cells showed a marked increase in tyrosine-uptake rate with elevated Vmax and unchanged Km.

Chronic hypertension increases tyrosine transport across the blood-brain barrier in the rat

We investigated the effect of chronic gradual blood pressure elevation on the brain uptake index (BUI) of the neutral amino acid tyrosine. We found a significant correlation between tyrosine's BUI and systolic blood pressure (r=0.580, p < 0.001) though changes of these two parameters were not parallel. Between 145 and 180 mmHg there was an abrupt elevation of BUI from an average 27% to 34%, which remained unchanged thereafter, even up to 290 mmHg. This suggests that a resetting rather than breakdown of the carrier transport mechanism may occur with gradual blood pressure rise above a certain level.

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na + gradient ([ Na +] outside > [ Na +] inside ). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide m- chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.

Sparing of the Brain in Neonatal Undernutrition: Amino Acid Transport and Incorporation into Brain and Muscle

Rates of tyrosine and lysine transport and incorporation into protein were measured in control and undernourished weanling rats. Undernutrition was induced by feeding lactating dams a low protein diet (12 percent casein) from birth to day 21. At weaning, body and brain weights of undernourished rats were 50 percent and 88 percent, respectively, of control values. Lysine and tyrosine transport rates into skeletal muscle were reduced by over 75 percent, more than twice the reduction seen in brain. Rates of amino acid incorporation into muscle protein were reduced by approximately 50 percent; the change in rate of incorporation into brain protein was not statistically significant. These data indicate that, in spite of marked retardation of amino acid transport into brain, the brain seems fully capable of maintaining normal rates of protein synthesis.

Transport of tryptophan and tyrosine in rat brain slices in the presence of lithium

Slices from rat cerebral cortex, brain stem, and cerebellum were incubated in media in which 1, 10, or 100 mmol/liter NaC1 had been replaced by equimolar amounts of LiC1. The initial influx of tryptophan and tyrosine into the slices diminished in the lithium-containing media. The lithium-induced inhibition was not competitive. The equilibrium accumulation of the amino acids was also less in the presence of LiC1. The incorporation of tryptophan and tyrosine into the proteins of the slices was inhibited by lithium. These were no clear differences between the brain areas studied. It has been suggested earlier that a lithium treatment enhances the in vivo cerebral uptake of these aromatic amino acids. The present results show that such a possible increase in uptake is not a direct effect of lithium ions on cell membranes.

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K(+) > Rb(+) > (Na(+) = Li(+)). Calcium and Mg(2+) ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO(4) (3-) > NO(3) (-) > Cl(-). The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca(2+) ions, or to effects on K(+) uptake. A mixture of Na(+) and K(+) in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na(+) plus K(+). The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.

Metabolic changes in Crithidia fasciculata accompanying physiological adaptation to growth in the presence of carbonyl cyanide [formula omitted

1. 1. Crithidia fasciculata adapted to growth in the presence of 10 −5 M carbonyl cyanide m- chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, maintained adenosine phosphate pools and an adenylate energy charge comparable to those of control cells. 2. 2. CCCP-adapted cells in the presence of the uncoupler respire endogenous substrate at a greater rate than control cells and this effect of CCCP appears readily reversible. 3. 3. CCCP-treated, adapted cells, supporting high endogenous respiration rates, were not responsive to added substrates which significantly stimulated the oxygen utilization of normal C. fasciculata. 4. 4. CCCP-adapted cells, provided with [U- 14C]-labeled proline, utilize this substrate at 67% the rate of control cells, but divide the isotopic label between CO 2 and protein in a ratio identical to that of normal cells. 5. 5. The transport of alamine and proline by adapted C. fasciculata was severely impaired, while the transport of tyrosine and leucine was unaffected.

Effect of gamma-glutamyl cycle inhibitors on brain amino acid transport and utilization.

Two inhibitors of the gamma-glutamyl cycle, methionine sulfoximine (MSO) and 2-imidazolidone-4-carboxylic acid (ICA) were administered to C57LB/6J mice. Both agents resulted in a reduced rate of transport of tyrosine from blood to brain and a decreased rate of incorporation of tyrosine from plasma into brain protein. MSO administration also diminished to concentrations of brain tyrosine, dopamine, and norepinephrine. MSO decreased the transport rate of valine by brain as well as the rate of its incorporation into protein when expressed in relation to the plasma specific activity. The results demonstrate a significant role for the gamma-glutamyl cycle in the transport of large neutral amino acids from blood to brain.

Selective changes in tyrosine transport by isolated choroid plexus exposed to inorganic lead

Freshly excised cat brain tissues were preincubated for 15 min in artificial cerebrospinal fluid (CSF) only or in CSF plus lead nitrate (5 × 10 −6 m) and then incubated for an additional 5 min with varying concentrations of l-[ 3H]tyrosine. The time course (from 5 to 60 min) for the accumulation of l-[ 3H]tyrosine showed the highest uptake in the choroid plexus (CP); the mean steady-state distribution ratio at 60 min was close to 15. This value exceeded by twofold the uptake by the neural tissues. The relationship between V net and the medium l-[ 3H]tyrosine concentration was a rectangular hyperbola. The apparent K m value for all three tissues was similar, but the V max of the CP was more than three times that of the neural tissues. Addition of lead nitrate altered the kinetics of uptake of l-[ 3H]tyrosine by the CP; the V max was reduced by one-half, whereas the affinity of carrier for the amino acid was approximately tripled. There was no effect on the transport of l-[ 3H]tyrosine by caudate nucleus or hypothalamus.

Transport of tyrosine, phenylalanine, tryptophan and glycine in neuroblastoma clones.

Abstract—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. Km and Vmax 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.

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.

Possible role of insulin in the transport of tyrosine and tryptophan from blood to brain

The administration of insulin or the ingestion of glucose increases the concentration of tryptophan and tyrosine in the brain. This increase is associated with a parallel decrease in the concentration of free tryptophan and tyrosine in serum. The results suggest that insulin enhances the transport of tyrosine and tryptophan from blood to brain.

Aromatic amino acid transport in Yersinia pestis

The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.

Independence of glucocorticosteroids and blood-brain barrier amino acid permeability

The effects of abnormal levels of circulating glucocorticosteroids upon the carrier-mediated transport of tyrosine and tryptophan across the blood-brain-carrier of the adult male rat were studied. The uptake of C 14 labeled tyrosine or tryptophan was measured relative to that of simultaneously injected 3H 2O, a freely diffusible internal standard. Fifteen sec after rapid intracarotid injection the rats were decapitated and brain tissue was homogenized and subjected to liquid scintillation counting. The level of circulating glucocorticosteroids was altered by intraperitoneal injection of cortisol (30 mg/kg) or corticosterone (30 mg/kg) 20 min prior to the amino acid injection. Rats were also injected with cortisol (10 mg/kg) or corticosterone (10 mg/kg) for 14 days. These rats were then injected with cortisol (10 mg/kg) or corticosterone (10 mg.kg) 90 min before the experiment. The amino acid uptake indices were analyzed statistically by the Wilcoxan Rank Sum Test. In no case was the uptake of tyrosine or tryptophan found to differ significantly ( p <0·10) between the experimental and control rats. It is concluded that no correlation between levels of circulating adrenal glucocorticosteroids and transport across the blood brain barrier of the amino acid precursors of the putative neurotransmitters, norepinephrine and serotonin, can be demonstrated by this method.

Transport synaptosomal du tryptophane et de la tyrosine cerebrale. Stimulation de la vitesse de capture apres reserpine ou inhibition de la monoamine oxydase

Transport of tryptophan (Trp) and tyrosine (Tyr) across synaptosomal membranes was studied in vitro after administration of reserpine or monoamine oxydase inhibitor (IMAO). A long-lasting stimulation of the high and low affinity uptake of the two aminoacids was observed in different brain structures. Moreover, no direct effect of these drugs was observed in vitro. Kinetic study of the high affinity systems showed different mechanisms of regulation for Trp and Tyr uptake after reserpine administration. According to these results the increase of cerebral amounts of Trp and Tyr after reserpine or IMAO treatment previously described could be induced by a stimulation of transport of these aminoacids by central nerve endings. However, mechanism or inducing factors of these regulations remain unknown.

Local and systemic effects of phenylalanine on intestinal transport of tyrosine and tryptophan.

SummaryThe local and the systemic effects of Phe on the intestinal transport of 1 mM Tyr and 5 mM Trp in Krebs-Henseleit buffers were studied in vitro by everted rat intestinal loops and in vivo by perfusion. The presence of equimolar Phe in the buffer did not inhibit the transport of Tyr and Trp in the in vitro everted intestinal loops, or in the in vivo perfusion studies. However, there was a significant inhibition in the in vitro and in the in vivo experiments (36 and 37%, respectively) when Phe was present in the buffers in a 10:1 ratio. Hyperphenylalanine-mia did not alter amino acid absorption. There were no differences in the transport of Tyr and Trp both in vitro and in vivo between the intestinal loop preparations of rats fed diets containing either 1.2, 8.2 or 0.1% Phe and 0.1% pCPA.

Molecular basis for the differential anti-metabolite action of D-tyrosine in strains 23 and 168 of Bacillus subtilis.

The basis for the difference between strains 168 (d-tyrosine-sensitive) and 23 (d-tyrosine-resistant) of Bacillus subtilis at the molecular level is that of transport of d-tyrosine into the cell. Strain 23 does not incorporate significant amounts of d-tyrosine into whole cells. A mutant derivative was isolated from strain 23 which had an altered transport system permitting d-tyrosine uptake, a change which also led to inhibition of growth by d-tyrosine. Strain 168 is extremely sensitive to growth inhibition caused by low concentrations of the d-isomer of tyrosine. A mutant derivative of strain 168 selected for its d-tyrosine resistant phenotype had an altered transport system which no longer recognized the d-isomer of tyrosine. These mutants define at least one element of the tyrosine transport system in B. subtilis and provide a convenient phenotype for the eventual location of the chromosal map position.