Formation and Fate of Tyrosine

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 52, Issue of December 25, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Formation and Fate of Tyrosine INTRACELLULAR PARTITIONING OF NEWLY SYNTHESIZED TYROSINE IN MAMMALIAN LIVER* (Received for publication, August 17, 1998, and in revised form, September 23, 1998) Ross Shiman and Douglas W. Gray From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 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. 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 , 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 Materials 2,6-[ring- 3 H]Phenylalanine, 2,6-[ring- 3 H]tyrosine, and * The costs of publication of this article were defrayed in part by the [ 35 S]methionine were purchased from Amersham Corp. The labeled payment of page charges. This article must therefore be hereby marked phenylalanine and tyrosine were repurified on an Aminex Q15S (Bio- advertisement in accordance with 18 U.S.C. Section 1734 solely to Rad) sulfonic acid ion exchange resin (17). AG-1 8 ( 400 mesh) and indicate this fact. AG-50 4( 400 mesh) were from Bio-Rad. Crystalline and fraction V To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology H171, The Pennsylvania State University bovine serum albumin (BSA), 1 all cell culture components, and Triton College of Medicine, Hershey, PA rshiman@psu.edu. Present address: Dept. of Health Evaluation Sciences, Pennsylvania State University College of Medicine, Hershey, PA The abbreviations used are: BSA, bovine serum albumin; [ 3 H]Tyr m, This paper is available on line at

2 Formation and Fate of Tyrosine in Hepatocytes 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- 3 H]phenylalanine was used rather than the [ 14 C]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 g 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% CO 2, 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 M linoleic acid, 66 M ethanolamine, 0.05 mg/ml gentamycin, trace metals, 160 M ascorbic acid, 15 mm Hepes, 0.1 M dexamethasone, 10 nm glucagon, 10 nm insulin, and amino acids to give final concentrations ( M): 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 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% CO 2, 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, [ 35 S]methionine and 2,6-([ring- 3 H]phenylalanine were added together to the culture medium (50 l, 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 [ 3 H]phenylalanine or [ 3 H]tyrosine specific radioactivity due to intracellular dilution by the respective unlabeled amino acid were measured by including 230 M [ 35 S]methionine in the culture media. This was about 5 times the normal rat plasma concentration (27) [ 3 H]Tyr P, and [ 3 H]Tyr degr are the rates at which [ 3 H]tyrosine was transported to the medium, incorporated into cell protein, and degraded, respectively; the sum of these is [ 3 H]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. and sufficient to swamp the intracellular methionine pool; further increases in concentration of [ 35 S]methionine had no measurable effect on 35 S label incorporation into protein. In our assay, changes in tyrosine or phenylalanine concentration had within experimental error ( 5%) no affect on [ 35 S]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 [ 3 H]tyrosine transported to the medium ([ 3 H]Tyr m ) and [ 3 H]tyrosine degradation products ([ 3 H]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 H 2 O) 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 HClO 4 (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). [ 3 H]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 [ 3 H]Tyr degr, 0.4 ml of the same supernatant (preceding paragraph) was added to a 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 H 2 0 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 [ 3 H]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 cm bed of AG1 8 resin (hydroxide form). After the sample had entered the bed, the column was washed with 1.0 ml of H 2 O. 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 CO 2, 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 3 H and 35 S Incorporation into Cell Protein For this, 300 l of culture extract were added to 50 l of BSA Fraction V (60 mg/ml H 2 O) containing 0.1 M methionine, 0.1 M phenylalanine, M tyrosine. (Frozen extract was thawed and sonicated, 3 times for 5 s/time (0 C).) After 10 min (0 C), 16 l of7n HClO 4 (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 HClO 4 (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, C), 10 ml of scintillation fluid (28) was added and the radioactivity measured in a scintillation counter. Quantitation of [ 3 H]Tyrosine and [ 3 H]Phenylalanine in Cell Protein For this, 0.5 ml of 0.1 M methionine, 0.1 M phenylalanine, M tyrosine in distilled H 2 O 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 ( 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 [ 3 H]tyrosine and

3 34762 Formation and Fate of Tyrosine in Hepatocytes FIG. 1.Effect of [ 3 H]phenylalanine concentration on rates of [ 3 H]tyrosine formation and degradation in hepatocytes. Shown are [ 3 H]Tyr total formed in picomole of Tyr/min/ g of DNA, and the fraction of [ 3 H]Tyr total that is degraded ([ 3 H]Tyr degr ), that is released as tritiated water (THO), and that is found in the medium (Tyr m ) versus [ 3 H]phenylalanine concentration of the culture medium. Tyrosine concentration in the medium was 250 M in all cases. Here and elsewhere for each condition tested, duplicate dishes were incubated, harvested, and assayed. All data are shown. [ 3 H]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 [ 3 H]tyrosine ([ 3 H]Tyr p ) and [ 3 H]phenylalanine ([ 3 H]Phe p ) in cell protein. When [ 3 H]tyrosine rather than [ 3 H]phenylalanine was used, the culture medium was assayed only for [ 3 H]tyrosine degradation products, and the 3 H/ 35 S 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 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- 3 H]phenylalanine to labeled products during the assay period. This rate ([ 3 H]Tyr total ) is the sum of the rates at which labeled tyrosine was exported to the medium ([ 3 H]Tyr m ), was degraded ([ 3 H]Tyr degr ), and was incorporated into cell protein ([ 3 H]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 [ 3 H]phenylalanine concentration increased the rate of [ 3 H]tyrosine formation about 100-fold, from 1 to 95 pmol/min/ g of DNA. Even at very high rates of tyrosine formation, only a minority ( 15%) of newly synthesized [ 3 H]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. 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 [ 3 H]phenylalanine almost completely ( 97%) blocked formation of [ 3 H]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 ([ 35 S]methionine incorporation) and by this criterion were not toxic to the cells. Effect of Exogenous, Unlabeled Tyrosine on the in Situ Fate of [ 2 H]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 [ 3 H]tyrosine formation from [ 3 H]phenylalanine or the partitioning of newly synthesized tyrosine between degradation and export ([ 3 H]Tyr degr /[ 3 H]Tyr m ratio) at either 50 or 130 M [ 3 H]phenylalanine (Fig. 2). The increase in tyrosine did dilute the intracellular [ 3 H]tyrosine pool, however, causing about a 3-fold decrease in [ 3 H]tyrosine incorporation into cell protein (Fig. 2). There was no effect ( 5%) of tyrosine concentration on the rate of protein synthesis. Complete omission of tyrosine from the medium had a dramatic effect on the relative amounts of [ 3 H]tyrosine that were degraded and incorporated into cell protein (Fig. 3B). With 0 M tyrosine and 50 M [ 3 H]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 M, 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 M [ 3 H]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 M 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). 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

4 Formation and Fate of Tyrosine in Hepatocytes 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 [ 3 H]phenylalanine (PHE). At the end of the assay period, cultures were harvested and [ 3 H]Tyr degr, [ 3 H]Tyr m, and [ 3 H]Tyr p were quantitated. From these, [ 3 H]Tyr total (pmol/min/ g of DNA) and the [ 3 H]Tyr degr /[ 3 H]Tyr m ratio were calculated. 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 The equations and their derivation are given in the Appendix. A rate constant of tyrosine transport, k 1, was calculated from the results in Fig. 5, A and B. The equations used (Equations 2 and 3) relate relative amounts of [ 3 H]tyrosine and [ 3 H]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 (k 3 ) and a 10-fold range of tyrosine concentrations. The k 1 (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, k 5, was calculated using Equation 1 and the results at a single phenylalanine concentration (i.e. a constant k 3 ). Results in Figs. 1 and 2 indicated that between 0.1 and 1 mm [ 3 H]phenylalanine, [ 3 H]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, k 4 /k ( 0.4) in this range. From this, the rate constant for tyrosine degradation, k 4, was calculated (Table I) under the assumption 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 [ 3 H]phenylalanine (PHE). At the end of the assay period, cultures were harvested and [ 3 H]Tyr degr,[ 3 H]Tyr m, and [ 3 H]Tyr p were quantitated. Results at 900 M (Panel A) and 50 M phenylalanine (Panel B) are shown. that the rate constants of tyrosine in-flow and out-flow, k 1 and k 2, were equal. Determination of [ 3 H]Tyrosine and [ 3 H]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 [ 3 H]tyrosine newly synthesized from [ 3 H]phenylalanine. The converse experiment was done in Fig. 6. Here, either unlabeled tyrosine from phenylalanine and protein degradation diluted [ 3 H]tyrosine from the medium, or unlabeled phenylalanine from protein degradation diluted [ 3 H]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 M in the experiment with [ 3 H]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. 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 k 1 (Table I), but was less than one-third as big as

5 34764 Formation and Fate of Tyrosine in Hepatocytes FIG. 4. Intracellular fate of [ 3 H]phenylalanine and [ 3 H]tyrosine derived from [ 3 H]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 k 1, k 2, and k 9, k 10 are apparent first-order rate constants for transport of tyrosine and phenylalanine between the medium and the respective protein precursor pools; k 5, k 6, and k 7, k 8 are apparent zero order rate constants for release and incorporation of tyrosine or phenylalanine from or into protein; and k 4 is an apparent first-order rate constant of tyrosine degradation. Although k 3 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. Tyr m, Tyr Phe *, and Tyr p are tyrosine that has been transported from the medium, derived from labeled phenylalanine, and released by proteolysis; Tyr degr * is radiolabel released by irreversible degradation of labeled tyrosine. Phe*, Phe m *, and Phe p 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. Tyr p * and Phe p * 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. k 1 k 2, 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. 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 [ 3 H]tyrosine. Likewise, Fig. 7 (open squares 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/ g of DNA using the conversion factors of 0.63 mg of protein/mg dry weight hepatocytes (30) and 16 g of DNA/mg of protein (present studies); from these, nanomole/min/ g ofdna nmol/h/mg dry weight/ The apparent first-order rate constant, k 1, calculated from Fig. 5A, is actually an average rate constant from 0 to 850 M 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 k (V m /K m ). Using this factor, the V m /K m in Table I gives a calculated apparent k 1 of 7.4( 1.6) 10 8 liter/min/ g of DNA, in reasonable agreement with the value of 5.9 ( 0.9) 10 8 liter/min/ g of DNA from Fig. 5 (Table I). FIG. 5.Effect of intracellular dilution on the specific radioactivity of [ 3 H]tyrosine formed from [ 3 H]phenylalanine. A, effect of tyrosine concentration on the [ 3 H]Phe p /[ 3 H]Tyr p 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 M phenylalanine, respectively. B, effect of [ 3 H]phenylalanine hydroxylation rate on the [ 3 H]Tyr p /[ 3 H]Phe p ratio in cell protein was plotted using Equation 3. The units on the x axis are (picomole of tyrosine formed per min/ g of DNA) 1. The tyrosine concentration for these measurements was 240 M. In this plot, the y intercept, the (Tyr/Phe) p ratio, has a value of Equivalent plots at other tyrosine concentrations (80, 160, 200, and 400 M) 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) 10 8 liter/min/ g of DNA at 50, 140, and 420 M phenylalanine, respectively. When multiplied by (Tyr/Phe) p, they give k 1 values of , 5.8.8, and liter/min/ g of DNA with an average k and circles) showed that an increase in medium phenylalanine from 0.16 to 0.8 mm had little effect on the degradation rate of [ 3 H]tyrosine from the medium despite a 6-fold increase in the rate of intracellular tyrosine formation. 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), [ 3 H]tyrosine derived from [ 3 H]phenylalanine was degraded about 2.5-fold faster than [ 3 H]tyrosine from the medium (Fig. 7). This finding was

6 Tyr k 1, k 2 b Formation and Fate of Tyrosine in Hepatocytes TABLE I Rate constants of phenylalanine and tyrosine metabolism Amino acid Rate constant Source a 5.9 ( 0.9) 10 8 liters/min/ g ofdna c Fig. 5, A and B 7.4 ( 1.6) 10 8 liters/min/ g of DNA Calculated from (V m /K M ) Tyr (footnote 3) (V m /K M ) Tyr 9.9 ( 2.1) 10 8 liters/min/ g of DNA Fig liters/min/ g of DNA Salter et al. (29) (footnote 2) k 4 38 ( 6) 10 8 liters/min/ g of DNA Fig. 5; Figs. 1 and 2, k 4 /k 2 6.5) k ( 1.6) pmol/min/ g of DNA Fig. 5 k ( 1.1) pmol/min/ g ofdna 2mM [ 3 H]Tyr in medium 5.3 ( 0.2) pmol/min/ g ofdna 1mM [ 3 H]Phe in medium 4.9 ( 0.5) pmol/min/ g of DNA From k 8 and (Tyr/Phe) p k 6 (rat liver) 4.2 ( 0.8) pmol/min/ g of DNA Calculated (footnote 6) Phe b k 8, k ( 0.5) pmol/min/ g ofdna 1mM [ 3 H]Phe in medium k 8 (rat liver) 7.2 ( 0.3) pmol/min/ g of DNA Calculated (footnote 6) k 9, k b 10 (V m /K M ) Phe 32 ( 3) 10 8 liters/min/ g of DNA Fig liters/min/ g of DNA Salter et al. (29) (footnote 2) Ratio (Tyr/Phe) p 0.64 ( 0.04) Fig. 5B a The source of the data or condition from which the constants were calculated. Where figure numbers are given, the equations used in the calculation are cited in the text. b The two rate constants are assumed to be equal. c Multiplying (liter/min/ g of DNA) ( g of DNA/liter), the DNA content of the hepatocytes (15), converts the units to min 1. FIG. 6. Effect of intracellular dilution on [ 3 H]phenylalanine and [ 3 H]tyrosine incorporation into cell protein. The upper line ( ) shows effects of [ 3 H]tyrosine concentration in the medium on the relative rate (R Tyr )of[ 3 H]tyrosine incorporation into cell protein; the phenylalanine concentration was 50 M. The lower line (E) shows effects of [ 3 H]phenylalanine concentration in the medium on the relative rate (R Phe ) of [ 3 H]phenylalanine incorporation into cell protein. Changes of tyrosine concentration in the medium from 80 to 420 M had no measurable affect on intracellular dilution of [ 3 H]phenylalanine. The data are plotted according to Equations 6 and 8 so that (V m /K m ) Phe k 7 /slope and (V m /K M ) Tyr (k 3 k 5 )/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/R Tyr or 1/R Phe axis equal to 1.0. Relative differences in rates of protein synthesis among cultures were corrected using [ 35 S]Met incorporation; changes in phenylalanine and tyrosine concentrations had no significant effect on the rate of [ 35 S]Met incorporation. Slopes for the [ 3 H]tyrosine and [ 3 H]phenylalanine lines were 42 ( 7) M and 22 ( 1.5) M, respectively. 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 FIG. 7.Rates of degradation of [ 3 H]tyrosine transported from the medium and formed from [ 3 H]phenylalanine. Shown are the effects of [ 3 H]tyrosine concentration in the medium on the in situ rate of degradation of [ 3 H]tyrosine at 0.16 mm ( ) and 0.8 mm phenylalanine (E), and the effects of the unlabeled tyrosine on the in situ rate of degradation of [ 3 H]tyrosine with 0.8 mm [ 3 H]phenylalanine in the culture medium (f). Tyrosine concentration in the medium is the x axis; the rate of [ 3 H]tyrosine degradation is the y axis. The solid line is calculated by fitting the data to Equation 10. The V m /K m values used were and liter/min/ g of DNA. The k 4 and K m values calculated for these values are in Table II. The solid line in the figure is for the value , but gave an identical fit, because within fairly broad limits, changes in V m /K m in Equation 10 can be compensated by changes in K m and k 4. The [ 3 H]Phe and [ 3 H]Tyr indicate the radiolabeled amino acid in the culture medium for the points defining the dashed and solid lines, respectively. 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 [ 3 H]tyrosine from the medium to the k 4 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,

7 34766 Formation and Fate of Tyrosine in Hepatocytes TABLE II Transport and degradation of exogenous tyrosine The K M(app) and k 4 values were calculated using Equation 10 to fit the tyrosine degradation data of Fig. 7 (solid circles) and the V m /K M values given in the table. (V m /K M ) Tyr K M(app) k 4 liter/min/ g DNA 10 8 mm liter/min/ g DNA a ( 1) 36. b ( 0.2) 0.94 c 37 ( 6) d a Tyrosine transport into pool used for protein synthesis (Table 1). b Tyrosine transport into whole cells (29). c This value corresponds to an apparent transport K M obtained from fitting the two transporter model of Salter et al. (29) to a single transporter model, see discussion preceding Equation 8 in Appendix. d Rate constant of degradation of newly synthesized tyrosine (Table I) or liter/min/ g of DNA, respectively (lines 1 and 2 in Table II). Whichever V m /K m was used, the calculated rate constant of tyrosine degradation, k 4, 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 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 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 [ 3 H]phenylalanine hydroxylation (Table III). Of particular interest, changes in the fractional rate of [ 3 H]tyrosine degradation, [ 3 H]Tyr degr /[ 3 H]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 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 M (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 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 g of DNA/dish with an intracellular volume of 15 l/dish (15), and had rate constants of tyrosine export (k 2 ) and degradation (k 4 ) of 6 and liter/min/ g of DNA, respectively (Table I). From these, the combined rate constant for tyrosine loss is about 1/min, corresponding to a tyrosine t1 2 of about 0.7 min. Since in the experiments cultures were in 0 M tyrosine medium

8 Formation and Fate of Tyrosine in Hepatocytes TABLE III Effect of hormones on tyrosine partitioning and tyrosine aminotransferase activity Twenty-five hours prior to assay, medium was removed from the cultures, the cultures were washed twice, 30 min each time, with 4.0 ml of standard medium containing insulin, insulin plus dexamethasone, or insulin plus dexamethasone plus glucagon. Twenty-four hours later, cultures were assayed in medium of the same composition as used in the 24-h incubation. When present in the medium, hormone concentrations were the same as in the standard medium. The phenylalanine concentration was 430 M at the time of addition of label. The rate of tyrosine synthesis ([ 3 H]Tyr total ) and fraction of the synthesized tyrosine degraded ([ 3 H]Tyr degr /[ 3 H]Tyr total ) were measured from culture medium obtained after 1 h incubation with [ 3 H]phenylalanine. Tyrosine aminotransferase activity (nanomole/min/ g of DNA) was measured in extracts of the same cultures to which label had been added. Hormones in medium Tyrosine amino transferase activity [ 3 H]Tyr degr /[ 3 H]Tyr total [ 3 H]Tyr total nmol/min/ g DNA (rel. %) ratio (rel. %) pmol/min/ g DNA Insulin dexamethasone glucagon (100) (100) 82 3 Insulin dexamethasone (41) (90) 73 1 Insulin (13) (70) 76 1 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. [ 35 S]methionine incorporated into cell protein at 0 and 250 M tyrosine were and , 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 tyrosyltrna 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 [ 3 H]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 M exogenous tyrosine about 65% of the tyrosine released by protein turnover was reincorporated into cell protein (2.8 of 4.3 pmol/min/ g DNA). This percentage is essentially identical to the percent of newly synthesized [ 3 H]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 [ 3 H]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 [ 3 H]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 tyrosyltrna/ g of DNA, which is only a few percent of the calculated intracellular free tyrosine pool at a normal plasma level of tyrosine (80 M (27)). At the rates of tyrosine incorporation measured here ( 5 pmol/min/ g 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 M (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. 5 R. Shiman and D. W. Gray, unpublished results.