J Biol Chem, Vol. 273, Issue 52, 34760-34769, December 25, 1998

Formation and Fate of Tyrosine
INTRACELLULAR PARTITIONING OF NEWLY SYNTHESIZED TYROSINE IN MAMMALIAN LIVER^*

Ross Shiman and Douglas W. Gray^§

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

	ABSTRACT

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Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.

	INTRODUCTION

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In animals, tyrosine is formed by hydroxylation of phenylalanine in a reaction catalyzed by phenylalanine hydroxylase (1, 2). The reaction occurs almost exclusively in liver (3), it is the first step in phenylalanine degradation (1, 4), and phenylalanine appears to be its primary regulator (5-7). Since liver is the site of tyrosine degradation as well as formation, the size of the intracellular tyrosine pool and the relative rates of tyrosine synthesis and degradation might be expected to depend, at least in part, on tyrosine concentration. Tyrosine could directly or indirectly (e.g. through the phenylalanine hydroxylase cofactor tetrahydrobiopterin (1, 8)) affect the rate of phenylalanine hydroxylation, it could affect the rate of tyrosine degradation, or it could affect the disposition (metabolic routing) of tyrosine in the cell. The mechanisms are not mutually exclusive, and all could operate, although a direct effect of tyrosine on phenylalanine hydroxylase has not, so far, been observed either in studies with purified enzyme or in the few in situ experiments that have addressed this question (9).

Historically, studies of regulation of the tyrosine pool have focused on tyrosine degradation, and most of the interest in degradation has been on control of tyrosine aminotransferase which catalyzes the first step in the pathway, deamination to p-hydroxyphenylpyruvate. The tyrosine aminotransferase reaction, although reversible, is usually considered the rate-limiting and regulated step in tyrosine metabolism (see Refs. 10-12, but see also Ref. 13), and the amount and activity of the enzyme appear largely, and possibly completely, hormonally controlled (10, 11). It is the next reaction, oxidation of p-hydroxyphenylpyruvate to homogentisate by p-hydroxyphenylpyruvate oxygenase (10), that is the first irreversible step in tyrosine degradation. If or how the activity of this enzyme or any enzyme in subsequent steps in the pathway are regulated is not clear. All the reactions, the hydroxylation of phenylalanine (14) and those in tyrosine oxidation (10), are catalyzed by cytoplasmic enzymes.

The present studies, which were directed at understanding the fate of newly synthesized tyrosine in liver, used primary rat hepatocytes maintained as nondividing, monolayer cultures as a liver model. The initial questions were to determine if and how tyrosine concentration affected tyrosine formation and distribution within the cell, and if tyrosine formed from phenylalanine had a fate different from tyrosine transported from the medium. The culture medium was defined and serum free; and in these cultures, rates of protein synthesis, urea synthesis, amounts and activity of phenylalanine hydroxylase and its cofactor tetrahydrobiopterin as well as the activity and hormone responsiveness of tyrosine aminotransferase are comparable to those in rat liver (15, 16). Although no evidence was found that tyrosine can regulate its own synthesis, the results did show that tyrosine from the medium and newly synthesized tyrosine are in different pools, that there appears to be metabolic channeling in tyrosine degradation, and that, under some conditions, the intracellular partitioning (the fate) of tyrosine in the cell is regulated. The studies provide a quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes which takes into account tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine incorporation into and release from cell protein.

	EXPERIMENTAL PROCEDURES

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Materials-- 2,6-[ring-³H]Phenylalanine, 2,6-[ring-³H]tyrosine, and [³⁵S]methionine were purchased from Amersham Corp. The labeled phenylalanine and tyrosine were repurified on an Aminex Q15S (Bio-Rad) sulfonic acid ion exchange resin (17). AG-1×8 (400 mesh) and AG-50×4(400 mesh) were from Bio-Rad. Crystalline and fraction V bovine serum albumin (BSA),¹ all cell culture components, and Triton X-114 for the scintillation mixture were purchased from Sigma. Water was deionized and glass distilled.

Assays for Tyrosine Aminotransferase, Phenylalanine Hydroxylase, Protein, and DNA-- Tyrosine aminotransferase (18) and phenylalanine hydroxylase (19) were assayed in fresh, sonicated culture extracts. (In the phenylalanine hydroxylase assay, 2,6-[ring-³H]phenylalanine was used rather than the [¹⁴C]phenylalanine cited in the reference.) Protein (20) and DNA (21) were determined on uncentrifuged, sonicated culture extracts; if culture extracts had been frozen, they were resonicated (3 times at 5 s/time) before taking samples for the determinations. Crystalline BSA and calf thymus DNA were the reference standards.

Curve Fitting and Data Analysis-- Data were fit to the given equations using Kaleidagraph (Synergy software). Errors for the calculated parameters are shown in parentheses. When the parameters came directly from curve fitting, they represent standard errors of the mean. In all other cases, they are standard deviations.

Hepatocyte Cultures and Standard Culture Medium-- Primary rat hepatocytes were prepared from male Sprague-Dawley rats by a modification (22) of the method of Berry and Friend (23). Procedure B of Kreamer et al. (24) was used to wash the cells except standard medium (defined below) was used in all centrifugations, and the final two centrifugations were at 21 °C. Cells (80 µg of DNA/collagen coated 60-mm dish) were seeded and maintained in standard culture medium (4.0 ml/dish). Medium was changed 4 h after seeding and every 24 h, thereafter. All culture incubations were at 37 °C in a 5% CO₂, 95% air atmosphere. "Standard hepatocyte culture medium," which is defined and serum-free, is a 1:1 mixture of Dulbecco's modified Eagle's medium (25) and Ham's F-12 medium (26) supplemented with 1 mg/ml bovine serum albumin (essentially fatty acid free), 0.1 mg/ml human transferrin (iron-free), 6.4 µM linoleic acid, 66 µM ethanolamine, 0.05 mg/ml gentamycin, trace metals, 160 µM ascorbic acid, 15 mM Hepes, 0.1 µM dexamethasone, 10 nM glucagon, 10 nM insulin, and amino acids to give final concentrations (µM): Ala-470, Asn-627, Asp-184, Arg-1320, Cys-100, Glu-75, Gly-3280, Gln-2666, His-313, Ile-531, Lys-2085, Leu-797, Met-233, Phe-538, Pro-1886, Ser-488, Thr-1079, Tyr-428, Val-866, and Trp-180 (16).

It takes 48-72 h after being put into culture for the hepatocyte cultures to exhibit in vivo levels of their differentiated activities; the cultures then appear stable through at least day 8 of culture (16). All data reported here were from experiments done on days 4, 5, or 6 of culture. Unless indicated otherwise, experiments were in situ; and except for specified changes in phenylalanine, tyrosine, or hormone concentrations the same (standard) culture medium was used in experiments and culture maintenance.

In Situ Assay of Phenylalanine Hydroxylation and Tyrosine Degradation-- For every experimental condition, duplicate hepatocyte cultures were harvested, assayed, and reported. All assay incubations were at 37 °C in a 5% CO₂, 95% air atmosphere. To measure phenylalanine hydroxylating activity in situ, cultures were preincubated (2 h) with continuous agitation (70 rpm on an orbital shaker) in standard medium with the phenylalanine, tyrosine, and hormone concentrations of the experiment. At the end of the 2-h period, [³⁵S]methionine and 2,6-([ring-³H]phenylalanine were added together to the culture medium (50 µl, pH 7) and the cultures immediately returned to the shaker and incubated for 1 h more. At the end of that time, the cultures were put on ice and the medium rapidly removed and saved at 0 °C. The cultures were then washed quickly 3 times with 4 ml (0 °C) of calcium, magnesium-free phosphate-buffered saline, following which 0.5 ml of harvesting buffer (0.02 M sodium phosphate, 0.4 M NaCl at pH 7.4 and 0 °C) was added to each dish and the cells scraped from the dish with a rubber policeman. (The dish was kept on ice at all times.) Addition of buffer (0.5 ml) and scraping was done two more times. The three samples were combined in a tared 2.0-ml microcentrifuge tube, weighed, and sonicated 3 times for 5 s/time at 0 °C. The extracts were then frozen in dry ice and stored at 80 °C. Tyrosine aminotransferase activity was determined prior to freezing the extracts.

Changes in [³H]phenylalanine or [³H]tyrosine specific radioactivity due to intracellular dilution by the respective unlabeled amino acid were measured by including 230 µM [³⁵S]methionine in the culture media. This was about 5 times the normal rat plasma concentration (27) and sufficient to swamp the intracellular methionine pool; further increases in concentration of [³⁵S]methionine had no measurable effect on ³⁵S label incorporation into protein. In our assay, changes in tyrosine or phenylalanine concentration had within experimental error (±5%) no affect on [³⁵S]methionine incorporation into cell protein. Control experiments showed that the high methionine concentration also had no effect on labeled tyrosine or phenylalanine incorporation.

Quantitation of Tyrosine and Tyrosine Degradation Products in the Culture Medium-- The [³H]tyrosine transported to the medium ([³H]Tyr_m) and [³H]tyrosine degradation products ([³H]Tyr_degr) were quantitated by analyzing the culture medium. (Less than 5% of either was found in the cell extracts.) To prepare medium for analysis, 0.9 ml of BSA Fraction V (15 mg/ml H₂O) was added with mixing to a 0.6-ml sample of culture medium in a 2-ml microcentrifuge tube. After 10 min at 0 °C, 0.15 ml of 7 N HClO₄ (0 °C) was added with mixing. After 10 more minutes at 0 °C, the mixture was centrifuged at 12,000 × g for 10 min (4 °C). [³H]Tyr_m was determined by adding 1.0 ml of this supernatant to 5.0 ml of 0.8 M HCl containing 0.36 M phenylalanine and 0.1 M tyrosine and labeled tyrosine quantitated by the isotope dilution method of Miller et al. (19).

To quantitate [³H]Tyr_degr, 0.4 ml of the same supernatant (preceding paragraph) was added to a 0.5 × 1-cm bed (in a Pasteur pipette plugged with a small amount of glass wool) of AG50×4 ion exchange resin (H⁺ form) equilibrated in water. After the sample entered the bed, 0.25, 0.25, and 0.1 ml of H₂0 were added in succession to the column (all at room temperature). The flow-through and successive eluates from the column were collected directly into a scintillation vial, 10 ml of scintillation fluid were then added and the radioactivity determined. Only labeled compounds containing an amino group adsorbed to this column, deaminated phenylalanine and tyrosine, and other labeled degradation products which did not bind to the resin constituted [³H]Tyr_degr.

The fraction of the labeled tyrosine degradation products present as tritiated water (THO) was determined by adding a 0.4-ml aliquot of the combined effluent from an AG-50 column (like that of the preceding paragraph) to a 0.5 × 1-cm bed of AG1×8 resin (hydroxide form). After the sample had entered the bed, the column was washed with 1.0 ml of H₂O. The flow-through and wash were collected in a scintillation vial, 10 ml of scintillation mixture were added and the sample counted. The rationale for this method was that up to the point of complete oxidation to CO₂, all tyrosine degradation products have a carboxyl group. Hence, tritium label not adsorbed by either the cation or anion exchanger was assumed to represent THO. Normally, at least 90% of the label came through this column.

Determination of ³H and ³⁵S Incorporation into Cell Protein-- For this, 300 µl of culture extract were added to 50 µl of BSA Fraction V (60 mg/ml H₂O) containing 0.1 M methionine, 0.1 M phenylalanine, 0.002 M tyrosine. (Frozen extract was thawed and sonicated, 3 times for 5 s/time (0 °C).) After 10 min (0 °C), 16 µl of 7 N HClO₄ (0 °C) containing 0.1 M methionine, 0.1 M phenylalanine, 0.04 M tyrosine was added and the mixture put on ice for 10 min. The mixture was then centrifuged at 2000 × g for 3 min (4 °C). The supernatant was discarded, and the precipitate was suspended with a vortex mixer in 2.0 ml of 0.3 N HClO₄ (0 °C) containing 0.1 M methionine, 0.1 M phenylalanine, and 0.04 M tyrosine; the mixture was centrifuged again at 2000 rpm for 3 min at 4 °C. This washing procedure was repeated one time more after which the supernatant was discarded and 0.64 ml of 0.1 N NaOH added. After the precipitate dissolved (overnight, 20-22 °C), 10 ml of scintillation fluid (28) was added and the radioactivity measured in a scintillation counter.

Quantitation of [³H]Tyrosine and [³H]Phenylalanine in Cell Protein-- For this, 0.5 ml of 0.1 M methionine, 0.1 M phenylalanine, 0.0015 M tyrosine in distilled H₂O was added to 0.5 ml of sonicated culture extract. After 20 min (0 °C), 0.17 ml of 70% trichloroacetic acid, at 0 °C, containing 0.1 M methionine, 0.1 M phenylalanine, 0.03 M tyrosine was added to the mixture. After 60 min (0 °C), sample were centrifuged at 3000 × g for 5 min at 4 °C. The precipitate was then washed and centrifuged 3 times with 1.0 ml (each time) of a 1:6 dilution of the above trichloroacetic acid/amino acid mixture. After the third wash, samples were extracted with 2 ml of ether to remove the trichloroacetic acid. The ether was then evaporated prior to hydrolysis. Samples were hydrolyzed with 0.5 ml of 6 N HCl in vacuo at 110 °C for 24 h. After drying the samples in vacuo (at 22 °C over NaOH pellets), 0.3 ml of 0.1 N HCl was added to each sample. Each sample was added to a column (0.5 × 4.5 cm) of Aminex (Bio-Rad) Q15S ion exchange resin equilibrated with 0.7 N HCl in 40% (v/v) ethanol. After the sample was applied, 0.25 ml of 0.1 N HCl was added to the column. The column was then developed with the equilibration buffer as described (17). In this system, tyrosine elutes before phenylalanine. The ratio of radioactivity in the [³H]tyrosine and [³H]phenylalanine peaks gave the ratio of these labeled amino acids in cell protein. This ratio and the total tritium in cell protein (see above) allowed calculation of [³H]tyrosine ([³H]Tyr_p) and [³H]phenylalanine ([³H]Phe_p) in cell protein.

When [³H]tyrosine rather than [³H]phenylalanine was used, the culture medium was assayed only for [³H]tyrosine degradation products, and the ³H/³⁵S ratio in cell protein was directly taken, without hydrolysis and chromatography, to calculate labeled tyrosine incorporation into cell protein.

Use of Pooled Data from Different Hepatocyte Preparations in Figs. 5 and 6-- The quantitative reproducibility of measurements from one set of hepatocyte cultures to the next made it practical to pool data from different experiments. This was done, specifically, in Figs. 5 and 6 to provide as many data points as possible. The pooled data were not specially selected; they were all data from all relevant experiments in which at least two determinations (in duplicate) had been done at significantly different concentrations of the experimental variable (phenylalanine or tyrosine).

Equations-- Equations and their derivations are given in the Appendix.

	RESULTS

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Effect of Phenylalanine Concentration on Tyrosine Formation and Fate-- The rate of tyrosine formation in primary rat hepatocytes was measured in situ from the conversion of 2,6-[ring-³H]phenylalanine to labeled products during the assay period. This rate ([³H]Tyr_total) is the sum of the rates at which labeled tyrosine was exported to the medium ([³H]Tyr_m), was degraded ([³H]Tyr_degr), and was incorporated into cell protein ([³H]Tyr_p). In the assay, tritium is only released when tyrosine is degraded: the first atom when p-hydroxyphenylpyruvate is oxidized to homogentisate, the second atom after complete oxidation of the carbon skeleton.

Tyrosine formation in hepatocytes is very responsive to phenylalanine concentration (9, 15). In Fig. 1, a 20-fold increase in [³H]phenylalanine concentration increased the rate of [³H]tyrosine formation about 100-fold, from 1 to 95 pmol/min/µg of DNA. Even at very high rates of tyrosine formation, only a minority (15%) of newly synthesized [³H]tyrosine was exported from the cells. The majority (70-90%) was degraded with about 90% of the tritium label of the degraded tyrosine being found as THO (Fig. 1), indicating that tyrosine degradation normally went to completion. Phenylalanine concentration had a relatively small effect on the fraction of tyrosine degraded or exported (Fig. 1), implying either that high rates of tyrosine formation did not saturate the tyrosine degradation and transport pathways, that rates of degradation and transport had similar dependence on tyrosine concentration, or both.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effect of [3H]phenylalanine concentration on rates of [3H]tyrosine formation and degradation in hepatocytes. Shown are [3H]Tyrtotal formed in picomole of Tyr/min/µg of DNA, and the fraction of [3H]Tyrtotal that is degraded ([3H]Tyrdegr), that is released as tritiated water (THO), and that is found in the medium (Tyrm) versus [3H]phenylalanine concentration of the culture medium. Tyrosine concentration in the medium was 250 µM in all cases. Here and elsewhere for each condition tested, duplicate dishes were incubated, harvested, and assayed. All data are shown.

In the standard assay, cultures were preincubated for 2 h in the experimental medium prior to addition of tracer amounts of labeled amino acids. Depending on its initial concentration, significant amounts (up to 20%) of phenylalanine and lesser amounts of tyrosine were catabolized by the cells during this period. Corrections for these decreases have been made in the graphs and tables. The corrections were calculated by assuming that the pseudo first-order rate constant for loss of amino acid during the 2-h preincubation was equal to the measured rate constant of amino acid loss during the assay incubation itself. For all experiments and conditions, duplicate cultures were assayed. Results from both cultures are shown.

Control experiments showed that the combined addition of specific inhibitors of tetrahydrobiopterin synthesis, 0.5 mM N-acetyl serotonin and 0.5 mM diaminopyrimidine (8), to standard medium containing 1 mM [³H]phenylalanine almost completely (>97%) blocked formation of [³H]tyrosine, tritiated degradation products, and THO in the hepatocytes. The result indicated that the only significant pathway for tritium label release required tetrahydrobiopterin, consistent with phenylalanine catabolism in the liver cells being dependent on phenylalanine hydroxylase activity (1, 4). At the concentrations used, the inhibitors had no measurable effect on the rate of protein synthesis ([³⁵S]methionine incorporation) and by this criterion were not toxic to the cells.

Effect of Exogenous, Unlabeled Tyrosine on the in Situ Fate of [²H]Tyrosine-- Increasing the tyrosine concentration of the medium, from a normal physiologic level to about 6 times that value, had relatively little effect on either the rate of [³H]tyrosine formation from [³H]phenylalanine or the partitioning of newly synthesized tyrosine between degradation and export ([³H]Tyr_degr/[³H]Tyr_m ratio) at either 50 or 130 µM [³H]phenylalanine (Fig. 2). The increase in tyrosine did dilute the intracellular [³H]tyrosine pool, however, causing about a 3-fold decrease in [³H]tyrosine incorporation into cell protein (Fig. 2). There was no effect (±5%) of tyrosine concentration on the rate of protein synthesis.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of tyrosine on rates of tyrosine synthesis and incorporation into protein, and on relative rates of tyrosine degradation and export. Hepatocyte cultures were assayed in situ at the indicated concentrations of tyrosine and [3H]phenylalanine (PHE). At the end of the assay period, cultures were harvested and [3H]Tyrdegr, [3H]Tyrm, and [3H]Tyrp were quantitated. From these, [3H]Tyrtotal (pmol/min/µg of DNA) and the [3H]Tyrdegr/[3H]Tyrm ratio were calculated.

Complete omission of tyrosine from the medium had a dramatic effect on the relative amounts of [³H]tyrosine that were degraded and incorporated into cell protein (Fig. 3B). With 0 µM tyrosine and 50 µM [³H]phenylalanine, the majority (~70%) of newly synthesized tyrosine was incorporated into cell protein, only a minority (~20%) was degraded. When tyrosine in the medium was increased to 250 µM, the situation was reversed, so that relatively little newly synthesized tyrosine was incorporated into protein; the majority was degraded (Fig. 3B). This effect was not evident at 900 µM [³H]phenylalanine (Fig. 3A). At this concentration, the rate of tyrosine formation was more than 20 times the rate of tyrosine incorporation into protein, effectively swamping the intracellular pool. Changes in tyrosine or phenylalanine concentration had no significant effect on the rate of protein synthesis at either 50 or 900 µM phenylalanine (not shown). Except, perhaps, at high, nonphysiological concentrations (>0.5 mM), there was also little effect of tyrosine on the rate of phenylalanine hydroxylation in the cells (Figs. 2 and 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of tyrosine and phenylalanine concentrations on the fate of newly synthesized tyrosine. Hepatocyte cultures were assayed in situ at the indicated concentrations of tyrosine and [3H]phenylalanine (PHE). At the end of the assay period, cultures were harvested and [3H]Tyrdegr, [3H]Tyrm, and [3H]Tyrp were quantitated. Results at 900 µM (Panel A) and 50 µM phenylalanine (Panel B) are shown.

Tyrosine Fate Determined from Intracellular Dilution of Newly Synthesized Tyrosine-- The results in Figs. 1-3 reflect effects of tyrosine and phenylalanine transport, tyrosine degradation, and tyrosine incorporation into and release from cell protein on the intracellular partitioning of newly synthesized tyrosine. Fig. 4 gives a model that accounts for these results. Rate constants in the model were calculated as described below using Equations 1-10. The equations and their derivation are given in the Appendix.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Intracellular fate of [3H]phenylalanine and [3H]tyrosine derived from [3H]phenylalanine in the medium. The dashed lines enclose intracellular pools of tyrosine and phenylalanine used for protein synthesis; it is assumed a pools contents are in rapid equilibrium. The k1, k2, and k9, k10 are apparent first-order rate constants for transport of tyrosine and phenylalanine between the medium and the respective protein precursor pools; k5, k6, and k7, k8 are apparent zero order rate constants for release and incorporation of tyrosine or phenylalanine from or into protein; and k4 is an apparent first-order rate constant of tyrosine degradation. Although k3 has the units of a zero order rate constant for tyrosine formation from phenylalanine, it is an empirical, complex constant that depends on the concentration of phenylalanine in the medium, the rate of phenylalanine transport, and the specific activity of phenylalanine hydroxylase in the cells; it is always measured. Tyrm, TyrPhe*, and Tyrp are tyrosine that has been transported from the medium, derived from labeled phenylalanine, and released by proteolysis; Tyrdegr* is radiolabel released by irreversible degradation of labeled tyrosine. Phe*, Phem*, and Phep are, respectively, labeled phenylalanine in the medium, labeled phenylalanine transported from the medium into the precursor pool, and unlabeled phenylalanine released by proteolysis into the precursor pool. Tyrp* and Phep* are the respective labeled amino acids incorporated into cell protein. Although they are shown as separate constants, from the properties of the transporters (29) and the (steady state) conditions of the experiment, it is assumed that forward and reverse apparent rate constants for a process are equal; i.e. k1 = k2, etc. Because the assay period is only 1 h, it is also assumed that the amount of incorporated labeled amino acid that is released due to protein turnover is relatively small and can be ignored.

A rate constant of tyrosine transport, k₁, was calculated from the results in Fig. 5, A and B. The equations used (Equations 2 and 3) relate relative amounts of [³H]tyrosine and [³H]phenylalanine incorporated into protein to rates of tyrosine transport, tyrosine release during protein turnover, and phenylalanine hydroxylation. Transport was treated as a first-order process in these calculations, because the tyrosine concentrations used in Fig. 5A were low relative to the K_m of the dominant tyrosine transporter in hepatocytes (29), and in this concentration range the data were not precise enough to distinguish a first-order from a saturable process. As shown (Fig. 5A), the results were consistent with the equation. At each phenylalanine concentration, the data appeared to describe a straight line, and, equally important, the slopes of the lines were not significantly different over a 60-fold range of phenylalanine hydroxylation rates (k₃) and a 10-fold range of tyrosine concentrations. The k₁ (Table I) was calculated from the average of the slopes of the lines in Fig. 5A and the total tyrosine to total phenylalanine ratio in cell protein, (Tyr)/Phe)_p, from Fig. 5B. The rate constant for release of unlabeled tyrosine from protein, k₅, was calculated using Equation 1 and the results at a single phenylalanine concentration (i.e. a constant k₃).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of intracellular dilution on the specific radioactivity of [3H]tyrosine formed from [3H]phenylalanine. A, effect of tyrosine concentration on the [3H]Phep/[3H]Tyrp ratio in cell protein was plotted using Equation 2. To display all data on the same graph, both ordinate and abscissa values were divided by 60 and 6 for 50 and 140 µM phenylalanine, respectively. B, effect of [3H]phenylalanine hydroxylation rate on the [3H]Tyrp/[3H]Phep ratio in cell protein was plotted using Equation 3. The units on the x axis are (picomole of tyrosine formed per min/µg of DNA)1. The tyrosine concentration for these measurements was 240 µM. In this plot, the y intercept, the (Tyr/Phe)p ratio, has a value of 0.64 ± 0.04. Equivalent plots at other tyrosine concentrations (80, 160, 200, and 400 µM) gave within error the same ratio. The slopes of the lines in panel A are 8.3 (±1.3), 9.0 (±1.0), and 10.0 (±1.3) × 108 liter/min/µg of DNA at 50, 140, and 420 µM phenylalanine, respectively. When multiplied by (Tyr/Phe)p, they give k1 values of 5.3 ± 1.0, 5.8 ±.8, and 6.5 ± 1.0 × 108 liter/min/µg of DNA with an average k1 = 5.9 ± 0.9.

Results in Figs. 1 and 2 indicated that between 0.1 and 1 mM [³H]phenylalanine, [³H]tyrosine synthesized from the phenylalanine was degraded 6.5 (±0.4) times faster than it was exported from cells. Therefore, in terms of Fig. 4, k₄/k₂ = 6.5 (±0.4) in this range. From this, the rate constant for tyrosine degradation, k₄, was calculated (Table I) under the assumption that the rate constants of tyrosine in-flow and out-flow, k₁ and k₂, were equal.

Determination of [³H]Tyrosine and [³H]Phenylalanine Transport Rates from Intracellular Amino Acid Dilution-- In the experiment in Fig. 5, A and B, unlabeled tyrosine from the medium and from protein degradation diluted [³H]tyrosine newly synthesized from [³H]phenylalanine. The converse experiment was done in Fig. 6. Here, either unlabeled tyrosine from phenylalanine and protein degradation diluted [³H]tyrosine from the medium, or unlabeled phenylalanine from protein degradation diluted [³H]phenylalanine from the medium. In these cases, the V_m/K_m values for transport of phenylalanine and tyrosine could be calculated (Equations 6 and 8) from the slopes of the lines in Fig. 6. The phenylalanine concentration was 50 µM in the experiment with [³H]tyrosine. At this concentration, the rate of tyrosine formation from phenylalanine, measured in parallel cultures, was less than one-third the rate at which tyrosine was released by protein degradation and could be taken into account with little error.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of intracellular dilution on [3H]phenylalanine and [3H]tyrosine incorporation into cell protein. The upper line () shows effects of [3H]tyrosine concentration in the medium on the relative rate (RTyr) of [3H]tyrosine incorporation into cell protein; the phenylalanine concentration was 50 µM. The lower line () shows effects of [3H]phenylalanine concentration in the medium on the relative rate (RPhe) of [3H]phenylalanine incorporation into cell protein. Changes of tyrosine concentration in the medium from 80 to 420 µM had no measurable affect on intracellular dilution of [3H]phenylalanine. The data are plotted according to Equations 6 and 8 so that (Vm/Km)Phe = k7/slope and (Vm/KM)Tyr = (k3 + k5)/slope. The slopes refer to the slopes in the plot for the appropriate line. Data have been normalized to make the y intercept on 1/RTyr or 1/RPhe axis equal to 1.0. Relative differences in rates of protein synthesis among cultures were corrected using [35S]Met incorporation; changes in phenylalanine and tyrosine concentrations had no significant effect on the rate of [35S]Met incorporation. Slopes for the [3H]tyrosine and [3H]phenylalanine lines were 42 (±7) µM and 22 (±1.5) µM, respectively.

The (V_m/K_m)_Phe for phenylalanine transport calculated from label dilution (Fig. 6, Table I) was in good agreement with the value from phenylalanine transport into the cell as a whole (Table I), indicating the processes measured by the two methods had a common rate-limiting step. The results with tyrosine were different. The (V_m/K_m)_Tyr for tyrosine transport calculated from Fig. 6 agreed reasonably well with the tyrosine transport rate constant k₁ (Table I), but was less than one-third as big as the (V_m/K_m)_Tyr for transport into the whole cell Table I.^2,3 That is, it appeared that tyrosine transport into the intracellular pool used for protein synthesis was slower than into the whole cell (Table I). This relative inability of extracellular tyrosine to compete with intracellular tyrosine as a source of the amino acid for protein synthesis implied the existence at least two tyrosine pools in the cells.

Intracellular Tyrosine Pools and Tyrosine Degradation in Hepatocytes-- Other results had hinted at there being more than one tyrosine pool. Figs. 2, 3, and 7 showed that an increase in medium tyrosine had little effect on the degradation rate of newly synthesized [³H]tyrosine. Likewise, Fig. 7 (open squares and circles) showed that an increase in medium phenylalanine from 0.16 to 0.8 mM had little effect on the degradation rate of [³H]tyrosine from the medium despite a 6-fold increase in the rate of intracellular tyrosine formation.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Rates of degradation of [3H]tyrosine transported from the medium and formed from [3H]phenylalanine. Shown are the effects of [3H]tyrosine concentration in the medium on the in situ rate of degradation of [3H]tyrosine at 0.16 mM () and 0.8 mM phenylalanine (), and the effects of the unlabeled tyrosine on the in situ rate of degradation of [3H]tyrosine with 0.8 mM [3H]phenylalanine in the culture medium (). Tyrosine concentration in the medium is the x axis; the rate of [3H]tyrosine degradation is the y axis. The solid line is calculated by fitting the data to Equation 10. The Vm/Km values used were 10 × 108 and 35 × 108 liter/min/µg of DNA. The k4 and Km values calculated for these values are in Table II. The solid line in the figure is for the value 35 × 108, but 10 × 108 gave an identical fit, because within fairly broad limits, changes in Vm/Km in Equation 10 can be compensated by changes in Km and k4. The [3H]Phe and [3H]Tyr indicate the radiolabeled amino acid in the culture medium for the points defining the dashed and solid lines, respectively.

The most convincing support for metabolically distinct tyrosine pools also came from results in Fig. 7. First, when compared at the same concentration (0.8 mM), [³H]tyrosine derived from [³H]phenylalanine was degraded about 2.5-fold faster than [³H]tyrosine from the medium (Fig. 7). This finding was unexpected, because phenylalanine is transported into the cell on the same transporter and at nearly the same rate as tyrosine (13, 29, 31). It must then be converted to tyrosine before it can be degraded. If anything, this predicts that tyrosine from phenylalanine should be degraded more slowly that tyrosine from the medium, the opposite of what was found.

The second supporting result came from the solid line in Fig. 7 which relates the rate of degradation of [³H]tyrosine from the medium to the k₄ of degradation and the K_m of transport. In the calculations, which used Equation 10, the (V_m/K_m)_Tyr was fixed either at the value for transport into the pool used for protein synthesis or at the value for transport into the cells as a whole, 9.9 × 10⁸ or 36 × 10⁸ liter/min/µg of DNA, respectively (lines 1 and 2 in Table II). Whichever V_m/K_m was used, the calculated rate constant of tyrosine degradation, k₄, was smaller for tyrosine from the medium (lines 1 and 2 in Table II) than for tyrosine synthesized from phenylalanine (line 4). Table II also shows that the V_m/K_m of 36 × 10⁸ gave a K_m(apparent) for tyrosine transport (line 2) in reasonable agreement with the K_m(apparent) for transport into the whole cell (line 3). In contrast, the V_m/K_m of 9.9 × 10⁸ gave a K_m(apparent) that was much larger than the reported value(s) (29), indicating that the (V_m/K_m)_Tyr for transport into the pool for protein synthesis did not provide a correct description of transport into the cell as a whole.

Effect of Tyrosine Aminotransferase Activity on the Fate of Newly Synthesized Tyrosine-- Dexamethasone, insulin, and glucagon are necessary for satisfactory maintenance of the hepatocytes. Glucagon and dexamethasone also cause an induction of tyrosine aminotransferase activity to levels about 10-fold higher than in the (uninduced) liver of a well fed rat. To determine the effect of tyrosine aminotransferase activity on intracellular partitioning of newly synthesized tyrosine, cultures maintained for 24 h in complete medium with or without glucagon and/or dexamethasone were compared (Table III). The absence of one or both hormones caused large decreases in tyrosine aminotransferase activity but had only small effects on the rate of [³H]phenylalanine hydroxylation (Table III). Of particular interest, changes in the fractional rate of [³H]tyrosine degradation, [³H]Tyr_degr/[³H]Tyr_total, were significantly smaller than changes in tyrosine aminotransferase activity. Hence, even with a relatively low level of tyrosine aminotransferase activity and a high phenylalanine hydroxylation rate, the majority of newly synthesized tyrosine was still degraded rather than exported.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The present work used primary rat hepatocytes in monolayer culture as a model system to examine effects of phenylalanine and tyrosine on formation and intracellular distribution of tyrosine in a liver cell. Rates of tyrosine synthesis, degradation, export, and incorporation into protein were measured as well as the intracellular dilution of the labeled tyrosine and phenylalanine incorporated into cell protein. We found that over a wide range of concentrations exogenous tyrosine had little effect on the rate of tyrosine formation from phenylalanine, which reinforced the idea that the role of phenylalanine hydroxylase is to degrade phenylalanine rather than to synthesize tyrosine, that the fate of the newly synthesized tyrosine could be accounted for by a kinetic scheme (Fig. 4) which took into consideration the significant in-flows and out-flows of tyrosine in the hepatocytes, and that tyrosine synthesized from phenylalanine and tyrosine transported from the medium are, at least partially, in different metabolic pools that are metabolized at different rates by the cells.

Although it would seem to make metabolic sense, tyrosine does not appear to regulate tyrosine synthesis. Up to now, except for experiments with primary hepatocytes in suspension culture (9), systematic studies under controlled conditions have been with purified phenylalanine hydroxylase. The in situ studies presented here, which tested a wider range of tyrosine and phenylalanine concentrations than heretofore, agree with the earlier results. The only effect of tyrosine we have found is a relatively small (25%) decrease in the rate of phenylalanine hydroxylation when phenylalanine concentration is low and tyrosine very high (Fig. 3). This effect could be due to competition for the transporter or have another source, but whatever its origin, it is only evident at >0.5 mM tyrosine (Fig. 2), well above the normal physiological range. Additional experiments, not shown but comparable to those presented, also showed little effect of tyrosine on phenylalanine hydroxylation. In these, we omitted culture hormones one at a time at total amino acid concentration of 1 and 10 times the normal rat plasma concentration. At each condition, tyrosine was also varied. In no case could we find evidence that in situ phenylalanine hydroxylase activity responded in a significant way to the size of the intracellular tyrosine pool.

Tyrosine also does not appear to regulate tyrosine aminotransferase activity. Early findings in vivo had suggested that tyrosine could induce the transaminase, but this effect was later shown to be secondary to hormone release under the stress of the tyrosine injections (11). In agreement, we also found no evidence that changes in the amino acid concentration of the culture medium affected transaminase activity measured in situ or in culture extracts (not shown). So far, the known significant regulators of the transaminase are either hormones or second messengers (10, 11), whose actions are to induce an increase in the amount of enzyme in the cell. It is generally assumed that through these effects the tyrosine aminotransferase reaction is the regulated and rate-limiting step in tyrosine degradation, although as Table III shows the latter is not true under all conditions.

A regulatory mechanism that has received little attention, but which at low tyrosine concentrations appears to be particularly effective, is that the distribution (partitioning) of tyrosine within the cell depends on tyrosine concentration. At normal plasma or higher concentrations of phenylalanine and tyrosine, only a relatively small fraction of newly synthesized tyrosine was exported from the hepatocytes, the majority was degraded; and the fractions exported or degraded were largely unaffected by the rate of phenylalanine hydroxylation or by the phenylalanine or tyrosine concentrations of the medium (Figs. 1-3). If the culture medium lacked tyrosine and phenylalanine was at a normal plasma concentration (50 µM (27)), relatively little newly synthesized tyrosine was transported to the medium or degraded, the majority was incorporated into protein. An interesting consequence was that the absence of tyrosine from the medium had little effect on protein synthesis, at least for the 3-h duration of the in situ assay.⁴ (Relative amounts of [³⁵S]methionine incorporated into cell protein at 0 and 250 µM tyrosine were 0.93 ± 0.03 and 1.00 ± 0.05, respectively.) Although some sort of intracellular compartmentation might be able to account for these results, a simpler mechanism is kinetic in which the rate of amino acid activation by tyrosyl-tRNA synthetase is faster than the rate of tyrosine degradation. Results in Fig. 3B require only that the rate of reaction of the tRNA synthetase with tyrosine be at least 2.5 times faster than the combined rates of [³H]tyrosine degradation and export. The amount and activity of tyrosyl-tRNA synthetase in rat liver is consistent with this possibility (32). Such a mechanism would be effective because there is only a limited amount of free synthetase and tRNA in a cell and aminoacylation and loading onto tRNA are effectively unidirectional.

If a kinetic mechanism involving rapid tRNA loading controls tyrosine distribution, one might also expect it to affect the fate of tyrosine released by protein turnover. The results in Fig. 3 support this possibility. It can be calculated from the data in the figure that at 0 µM exogenous tyrosine about 65% of the tyrosine released by protein turnover was reincorporated into cell protein (2.8 of 4.3 pmol/min/µg DNA). This percentage is essentially identical to the percent of newly synthesized [³H]tyrosine incorporated into protein under the same conditions, indicating that tyrosine from the two sources had quantitatively the same fate. This kinetic mechanism involving rates of tRNA loading provides an unsuspected (partial) answer to the problem of how effects of tyrosine depletion on cell viability are minimized. The efficiency of the mechanism in conserving tyrosine at low concentrations of exogenous tyrosine is striking, since tyrosine aminotransferase and tyrosine degradation were fully induced in these experiments.

Relative to the rate of protein synthesis, there is little aminoacylated tRNA in liver. In the case of leucine, even with a high concentration of the amino acid (10 times the normal plasma value), the pool of [³H]leucyl-tRNA in perfused rat liver is completely incorporated into nascent protein within 2 min (33). Since this includes the time required to chase free [³H]leucine from the tissue, the actual rate of turnover must be even faster. Aminoacyl-tRNA turnover in the cultured hepatocytes also appears rapid. Assuming a content similar to that in rat liver (33), hepatocytes will have about 0.5 pmol of tyrosyl-tRNA/µg of DNA, which is only a few percent of the calculated intracellular free tyrosine pool at a normal plasma level of tyrosine (80 µM (27)). At the rates of tyrosine incorporation measured here (~5 pmol/min/µg of DNA (Table I)), the pool of tyrosyl-tRNA in an hepatocyte would undergo essentially complete exchange in less than a minute.

Differences in degradation rates of exogenous (plasma) tyrosine and of tyrosine from phenylalanine have been reported before (34). The experiments were with rats infused at a constant rate with labeled phenylalanine or tyrosine at either a normal or an ~8 times normal concentration of phenylalanine. The authors found that only a minority of the tyrosine synthesized from phenylalanine appeared in the plasma (20 and 30% at normal and 8 times normal phenylalanine level, respectively), and inferred that tyrosine formed from phenylalanine was oxidized 2-3 times faster than tyrosine from the plasma. The present more direct and detailed studies in the primary hepatocytes support and extend these results.

The origin of this difference in rates of degradation is of considerable interest. It could be due to differences in rates of transport of the two amino acids or to newly formed tyrosine being more accessible to the tyrosine catabolic enzymes than tyrosine from the medium. The first possibility seems unlikely, because, in bulk, phenylalanine and tyrosine are transported at essentially the same rate (13, 29, 31) on the same transporter system in hepatocytes, the L system (35). Furthermore, when leucine concentration is constant, as it is in our experiments, the total activity of the L transport system in hepatocytes does not appear to significantly vary (35). The second possibility that newly synthesized tyrosine is more accessible to the tyrosine degradative enzymes than tyrosine from the plasma, that is that some form of metabolic channeling (36) is involved in degradation of tyrosine derived from phenylalanine, is consistent with all observations. Metabolic channeling in degradation of newly synthesized tyrosine implies the existence of at least two tyrosine pools with different kinetic properties. It could involve intracellular compartmentation or a physical association (proximity) of phenylalanine hydroxylase with enzymes involved in the initial steps of tyrosine degradation. The major argument against compartmentation is that phenylalanine hydroxylase (14) and the tyrosine catabolic enzymes (10) are soluble, cytoplasmic proteins; the alternative, physical association of some of the enzymes, has yet to be tested.

Whether the inability of exogenous tyrosine to decrease the degradation rate of newly synthesized tyrosine and of newly synthesized tyrosine to decrease the degradation rate of exogenous tyrosine are related to metabolic channeling is not clear, because they are also consistent with the degradative enzymes having high K_m values. When -ketoglutarate is saturating, tyrosine aminotransferase does have a high K_m,Tyr (~1.4 mM (37)), but p-hydroxyphenylpyruvate dioxygenase which catalyzes the next reaction, the first irreversible step in tyrosine degradation, does not; its K_m is 10-25 µM (38).⁵ The net effect is difficult to predict, because the equilibrium position and the apparent K_m of the transaminase depends on the -ketoglutarate/glutamate ratio and, hence, on the cell's metabolic status. The effect of changing this ratio was tested by addition of NH₄Cl to the culture medium to decrease, through the action of glutamate dehydrogenase, the -ketoglutarate concentration in the cells. In glucagon-free medium, 10 mM NH₄Cl decreased the tyrosine degradation rate by 75% with a concurrent, corresponding increase in tyrosine transport from the cells (data not shown). Thus, a decrease in the -ketoglutarate level changed the partitioning of tyrosine between export and degradation in an expected way, but only when tyrosine aminotransferase activity was relatively low. When glucagon was present and the transaminase induced, there was no effect of the NH₄Cl on the partitioning of tyrosine, implying another step in the pathway was rate-limiting under these conditions. Normal plasma levels of tyrosine and phenylalanine (80 and 50 µM, respectively) were used in these experiments, and neither the NH₄Cl nor glucagon concentrations affected the rates of phenylalanine hydroxylation or protein synthesis.

The present results provide an outline of tyrosine and phenylalanine utilization and distribution in hepatocytes that is summarized in Fig. 4. Despite its simplifications, the model and calculated rate constants have been quite successful in accounting for our results. This is exemplified in Fig. 5A where the effects of a large range of tyrosine and phenylalanine concentrations and phenylalanine hydroxylase activities are fit quite well by the model. It is also shown in the consistency of the rate constants. For k₁ and k₆, where it was possible to obtain the rate constants by independent methods, the different values agree within error (Table I). There is remarkably good agreement, as well, between the rates of tyrosine and phenylalanine incorporation into hepatocyte protein (k₆ and k₈) and in their respective rates of incorporation into rat liver protein (Table I).⁶ Finally, the rates of tyrosine incorporation and release from cell protein (k₆ and k₅ (Table I)) also agree within error, as would be predicted under the conditions of the experiments. The only caveat for some of the constants (k₆, k₈, and V_m/K_m) is that secreted protein was not taken into account when considering rates of amino acid incorporation into total protein. In consequence, rates of total protein synthesis are slightly underestimated. For the constants affected, the error is fairly small (10%),⁷ which in most cases is less than the experimental errors in the values of the constants. Constants calculated using the phenylalanine/tyrosine ratio incorporated into protein are not affected by this problem. Thus far, results of all experiments have been consistent with the model.

Several rate constants were obtained by measuring intracellular dilution of a labeled amino acid. This is an indirect method for studying the kinetics of amino acid transport and use, and its precision is limited. Nonetheless, it had distinct advantages for the questions we were asking. It measures transport into a specific amino acid pool, the one used for protein synthesis. It has allowed us to distinguish intracellular contributions of tyrosine from plasma, protein, and phenylalanine, to study in a controlled way the fate of an amino acid formed inside the cell, and to make a direct comparison of its rates of export, degradation, and incorporation into protein. The results have raised the possibilities of a kinetic control of tyrosine use and of a physical association of phenylalanine hydroxylase and enzymes of the tyrosine degradation pathway.

	ACKNOWLEDGEMENT

We thank Dr. Leonard Jefferson (Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine) for help in the preparation of the rat hepatocytes used in these experiments.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology H171, The Pennsylvania State University College of Medicine, Hershey, PA 17033. E-mail: rshiman{at}psu.edu.

^§ Present address: Dept. of Health Evaluation Sciences, Pennsylvania State University College of Medicine, Hershey, PA 17033.

The abbreviations used are: BSA, bovine serum albumin; [³H]Tyr_m, [³H]Tyr_P, and [³H]Tyr_degr are the rates at which [³H]tyrosine was transported to the medium, incorporated into cell protein, and degraded, respectively; the sum of these is [³H]Tyr_total, the total rate of tyrosine formation; Tyr_m, Tyr_p, and Tyr_Phe are unlabeled tyrosine from the medium, protein, and phenylalanine hydroxylation, respectively; asterisk (*), implies the presence of unspecified label in the compound.

² The published (29) V_m values for transport are given as nanomole of tyrosine/h/mg dry weight of hepatocytes. These numbers have been converted to nanomole/min/µg of DNA using the conversion factors of 0.63 mg of protein/mg dry weight hepatocytes (30) and 16 µg of DNA/mg of protein (present studies); from these, nanomole/min/µg of DNA = nmol/h/mg dry weight/600.

³ The apparent first-order rate constant, k₁, calculated from Fig. 5A, is actually an average rate constant from 0 to 850 µM tyrosine (0 to ~0.45 times the K_m) on a hyperbolic saturation curve. The relationship between this average constant and a V_m/K_m, calculated from the integrated, steady-state equation over the same concentration range, is k₁  0.75 × (V_m/K_m). Using this factor, the V_m/K_m in Table I gives a calculated apparent k₁ of 7.4(±1.6) × 10⁸ liter/min/µg of DNA, in reasonable agreement with the value of 5.9 (±0.9) × 10⁸ liter/min/µg of DNA from Fig. 5 (Table I).

⁴ As the following calculations show, when cultures were shifted to a zero tyrosine medium, there was only enough free tyrosine in the cytoplasm to sustain protein synthesis for a few minutes. An average hepatocyte culture contained approximately 40 µg of DNA/dish with an intracellular volume of 15 µl/dish (15), and had rate constants of tyrosine export (k₂) and degradation (k₄) of 6 and 35 × 10⁸ liter/min/µg of DNA, respectively (Table I). From these, the combined rate constant for tyrosine loss is about 1/min, corresponding to a tyrosine t_1/2 of about 0.7 min. Since in the experiments cultures were in 0 µM tyrosine medium for 120 min prior to addition of tracer amounts of labeled amino acids (see "Experimental Procedures"), there should have been no intracellular tyrosine remaining that had been carried over from the medium.

⁵ R. Shiman and D. W. Gray, unpublished results.

⁶ The rates of incorporation of tyrosine and phenylalanine into rat liver protein are 4.2 ± 0.8 and 7.2 ± 0.3 pmol/min/µg of DNA, respectively. These were calculated from the rate of protein synthesis in perfused rat liver of 4 mg of protein/h/g liver (33), the protein content of rat liver (200 mg/g liver (33)), the tyrosine and phenylalanine contents of rat liver protein (188 ± 29 and 349 ± 9 nmol/mg of protein (39)), and the protein to dna ratio of the hepatocyte preparations (16 µg of DNA/mg of protein).

⁷ In the experiments, the total labeling period is 1 h. Since secreted protein accounts for <20% of total protein synthesis, and it takes ~30 min from the time of label addition before significant labeled protein appears in the medium (data not shown), at most 10% of label incorporated is not taken into account in our calculations.

	APPENDIX

Equations for Calculating Rate Constants

Effects of Tyrosine Concentration and Phenylalanine Hydroxylation Rate on Relative Amounts of [³H]Tyrosine and [³H]Phenylalanine Incorporated into Cell Protein in Fig. 5-- Equation 1 was derived using the rate constants and model in Fig. 4. It was assumed that the intracellular tyrosine pool was in a steady state (assured by the assay conditions), that in this steady state rates of protein synthesis and degradation were, within error, equal to each other and to the rates of amino acid incorporation into cell protein during the 1-h labeling period, and that transporter saturation could be ignored, allowing tyrosine transport to be approximated as a first-order process. The justification for this last assumption is that the highest tyrosine concentration used in Fig. 5A, 0.8 mM, was much less than the 2 mM K_m of the dominant tyrosine transporter in hepatocytes (29), and the experimental data were not precise enough to distinguish a first-order from a saturable process in this region of the saturation curve.

In the model (Fig. 4), the tyrosine pool contains labeled tyrosine (Tyr_Phe*) synthesized from labeled phenylalanine (Phe_m*), unlabeled tyrosine transported from the medium (Tyr_m), and unlabeled tyrosine released by protein degradation (Tyr_p). The fraction of tyrosine that is labeled in the intracellular pool is (Tyr_Phe*/(Tyr_m + Tyr_p + Tyr_Phe*)). Since under steady-state conditions, the relative amount of each tyrosine species in the pool is proportional to its rate of in-flow, this fraction can be written k₃/(k₁(Tyr) + k₅ + k₃), where k₃ and k₅ are zero order rate constants of tyrosine formation and tyrosine release and k₁(Tyr) is the rate of transport of tyrosine from the medium into the pool. The fraction of tyrosine that is labeled is also equal to the ratio of labeled tyrosine and labeled phenylalanine incorporated into cell protein (Tyr_p*/Phe_p*) divided by the total tyrosine to total phenylalanine ratio in cell protein, (Tyr/Phe)_p. Combining the terms and rearranging leads to,

(Eq. 1)

Transformations of this equation, Equations 2 and 3, were used to plot the data in Fig. 5, A and B, respectively,

(Eq. 2)

(Eq. 3)

Although treated as a constant, k₃, the rate of tyrosine formation, is a function of phenylalanine concentration. As a result, each point in a data set used to calculate a line must be determined at the same phenylalanine concentration. The Tyr_p*/Phe_p* ratio (or its inverse) in these equations is unaffected by dilution of the [³H]phenylalanine precursor as long as the intracellular phenylalanine used for protein synthesis and for phenylalanine hydroxylation have similar specific radioactivities.

Determination of V_m/K_m of Phenylalanine and Tyrosine Transport from Intracellular Dilution of Transported Label in Fig. 6-- V_m/K_m values for phenylalanine and tyrosine transport into the pool used for protein synthesis were calculated from intracellular dilution of added labeled amino acid. The formulas for the calculations were derived from the model and rate constants in Fig. 4. In the derivation, phenylalanine and tyrosine transport were assumed to obey hyperbolic kinetics (29). The rate of incorporation of labeled phenylalanine into protein, R_Phe, is then,

(Eq. 4)

and in rearranged form,

(Eq. 5)

[Phe_m*] and [Phe_p] are the concentrations in the intracellular pool of labeled phenylalanine from the medium and unlabeled phenylalanine from protein degradation; k₈ is the rate constant of phenylalanine incorporation into protein. Since under steady state conditions the [Phe_p]/[Phe_m*] ratio in Equation 5 is equal to the ratio of rates at which the species enter the pool, k₇ can be substituted for [Phe_p] and, assuming hyperbolic kinetics, V_m/(K_M/[Phe*] + 1) for [Phe_m*]. [Phe*] is the concentration of labeled phenylalanine in the medium. Since V_m,transport k₇ in these cells (see "Results"), then,

(Eq. 6)

A comparable derivation can be made with tyrosine as the labeled amino acid. In this case, tyrosine dilution is from two sources: proteolysis (Tyr_p) and phenylalanine hydroxylation (Tyr_Phe). This gives,

(Eq. 7)

At 50 µM phenylalanine, the concentration in Fig. 6 when [³H]tyrosine was varied, k₃ < k₅. As long as V_m,transport k₃ + k₅ as shown under "Results," then,

(Eq. 8)

Relationship of Tyrosine Degradation and Transport in Fig. 7-- Degradation of exogenous tyrosine in Fig. 7 (open squares) was analyzed using Equation 10. The equation was derived from the model in Fig. 4. The derivation assumed tyrosine transport was