Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport.

Mutants of CHO-K1 cells with varied levels of A system activity, probably the result of increases in absolute amount of the A system transporter, have corresponding increases in levels of peptides banding at 62-66 and 29 kDa. Mutant alar4-H3.9, showing the highest increase of A system activity and of 62- to 66- and 29-kDa peptides, was selected for this study. The N terminus 16-amino acid sequence of the 62- to 66-kDa peptide(s) of this mutant showed between 80% and 100% identity with the mammalian mitochondrial 60-kDa heat shock protein P1 (hsp60). Two-dimensional gel electrophoresis of the 62- to 66-kDa band showed two major, a minor, and several smaller spots (of same mass but different pI values) for both wild type (WT) and mutant, with the two major spots being of greater density in the mutant. Immunoblots with antibody to P1 identified the two major and minor peptides as P1 related. Two-dimensional gels of whole cell extracts of the WT and alar4-H3.9 confirmed these findings and indicated that the two major bands of the mutant were 2.4 times as abundant as that found for the WT. A plasma membrane fraction of the mutant, exhibiting 4.8 times more A system activity than the WT, contained 3.6 times as much P1 as the WT. Immunoblots with antibodies to P1, mitochondrial malate dehydrogenase, and to the mitochondrial F1/F0-ATPase demonstrated that the increased amount of P1 observed in the mutant was not the result of increases in amount of mitochondrial protein. Northern blot analysis demonstrated that the mutant had 2.5 times as much mRNA for P1 as the WT. The close analogy with the relationship between A system and Na+,K(+)-ATPase suggests that there is a coordinate regulation of the A system of amino acid transport, Na+,K(+)-ATPase, and P1 protein, probably as a result of mutation in a shared regulatory element. The possible role of P1 in A system function is discussed.

The competence progression model in CHO-K1 cells: the relationship between protein kinase C and immediate early gene expression in the insulin mitogenic signal.

CHO-K1 cells grow in a defined medium with insulin, at physiological concentrations, as the only hormone. IGF-I can substitute for insulin. Quiescent cells require a 9-10-h lag, subsequent to the addition of insulin, to synthesize DNA. The phorbol ester, 12-O-tetradeconoylphorbol 13-acetate (TPA), cannot support growth of these cells, is a more effective inducer than insulin of c-fos, c-myc, c-jun, jun-B, Krox-20, Krox 24, fra-1 and JE, and induces fra-1, JE and c-myc with different kinetics from those of insulin. The addition of insulin + TPA to quiescent cells produces a synergistic effect on DNA synthesis but not on the expression of immediate early genes. Pretreatment of these cells with TPA or insulin decreases the required lag time for DNA synthesis by 3 h in a protein-synthesis-independent manner. These results, together with other experiments, demonstrate that [1] the insulin signal is independent of PKC, [2] insulin acts as a weak competence and a strong progression factor, while TPA behaves as a strong competence factor, and [3] the 9-10-h lag is made up of a 3-h period which is independent of protein synthesis, advancing the cells to a post-G(o) state of 'competence'.

Analysis of the genes involved in the insulin transmembrane mitogenic signal in Chinese hamster ovary cells, CHO-K1, utilizing insulin-independent mutants.

CHO-K1 cells, wild type (WT), grow in a defined medium with insulin as the only essential hormone. When starved for insulin, these cells accumulate in G0/G1 stage. Insulin binding to its receptor stimulates DNA synthesis and cell division and induces an increase in abundance of mRNA for c-fos, c-jun, Krox-20, Krox-24 (zif/268), fra-1, jun-B, c-myc, and JE. The kinetics of induction of these genes are similar to that shown with serum induction of 3T3. These genes show maximum stimulation at insulin concentrations of 20, 160, or 320 ng/ml and their expression is inhibited at higher concentrations. The addition of cycloheximide results in superinduction. The WT and insulin-independent mutants show no detectable signal for KC, fos-b, or nur77 and no increase over the basal level of pI-15, probably eliminating these genes as participants in the insulin mitogenic signal. These mutants synthesize DNA in the absence of insulin at rates that vary from 4 to 12 times that of the quiescent (insulin unstimulated) WT and are further inducible by insulin. The mutants have "constitutive" levels of Krox-24 (zif/268), fra-1, jun-B, c-myc, and JE (INS-type 2 genes) mRNAs that vary from mutant to mutant, reaching a maximum of an 8-fold increase for fra-1 and JE over the quiescent WT levels. There were no detectable levels of mRNA for genes c-fos and Krox-20 and no increase in level of mRNA for c-jun (INS-type 1 genes) as compared to the quiescent WT. Thus, although these INS-type 1 and type 2 genes may be involved in the full insulin mitogenic signal, the constitutive up-regulation of only genes in INS-type 2 is sufficient for insulin-independent DNA synthesis and cell division. Analysis of hybrids constructed between WT and mutant 27 indicate that the mutant phenotype is recessive, pointing to the existence of a regulatory gene producing a negative regulator.

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Previous work suggested that the structural gene for the A system transporter and the mRNA for the alpha subunit of the Na+,K(+)-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance of the alpha 1 subunit of Na+,K(+)-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K(+)-ATPase mRNA alpha 1-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K(+)-ATPase mRNA alpha 1-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-alpha-aminoisobutyric acid and beta-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K(+)-ATPase mRNA alpha 1-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and alar4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K(+)-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K(+)-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K(+)-ATPase.

Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes.

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone. The degree of expression of the mammalian System A activity in Xenopus oocytes corresponded to the known transport rates in the tissue from which the mRNA was prepared. For example, hepatic mRNA from glucagon-treated rats produced greater System A activity than mRNA from control animals, and the mRNA from the CHO transport mutant cell line alar4-H3.9, which overproduces System A, resulted in higher transport rates than mRNA from the parental cell line (CHO-K1). Fractionation of total mRNA poly(A+) by nondenaturing agarose gel electrophoresis revealed transport activity associated with a 2.0-2.5-kilobase mRNA fraction common to each of the three tissues tested.

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Insulin is the only hormone required for continued growth of Chinese hamster ovary CHO-K1 cells in the defined medium M-F12. When CHO-K1 cells are incubated in M-F12 without insulin for 48-72 hr, the cells accumulate in G1. In response to physiological concentrations of insulin an 18-fold increase in rate of DNA synthesis occurs due to cells entering S phase after an 8- to 10-hr lag; cell division begins after 24 hr. The inhibitory effect of actinomycin D and 5,6-dichlorobenzimidazole riboside indicates that RNA synthesis is required for progression to S phase. CHO-K1 cells possess insulin receptors, and the insulin effect results from insulin binding to its own receptor: (i) Binding occurs at physiological insulin concentrations with a half-maximal stimulation at approximately 14 ng/ml. (ii) At insulin concentrations used, insulin-like growth factor I and II (IGF-I and IGF-II) have little or no effect. (iii) Scatchard analysis of 125I-labeled insulin binding shows the curvilinear response typical of insulin. (iv) The Kd for the so-called high-affinity binding site and the Ke are characteristic of the insulin receptor. (v) At the minimal insulin concentrations that stimulate growth, IGF-I and IGF-II compete poorly with insulin for insulin binding, insulin competes poorly with IGF-I for IGF-I binding, and affinity labeling with 125I-labeled insulin identifies a polypeptide (Mr = 125,000) typical of the alpha subunit of the insulin receptor.

alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

A constitutive mutant, alar4, for the A system of amino acid transport, has increased activity and amount of the A system. This is accompanied by increased sensitivity to ouabain, as measured by efficiency of plating, and increased activity and abundance of the Na+,K+-ATPase that is present in the parental cell line, CHO-K1 (wild type). The latter was shown by increases in (i) ouabain-inhibitable 86Rb uptake in intact cells, (ii) ouabain-inhibitable ATPase activity in mixed membrane vesicles, and (iii) number of ouabain-binding sites and by similar Kd values for ouabain binding and K1/2 for ouabain inhibition of Na+,K+-ATPase as compared to the wild type. The increase in abundance of the Na+ pump is associated with a 4-fold increase in abundance of the mRNA for the alpha 1 subunit of the Na+,K+-ATPase. We could not detect mRNA for alpha 2 or alpha 3 or for the beta subunits. The increase in abundance of the A system and Na+,K+-ATPase is associated with a negligible increase in intracellular Na+ concentration. We propose that the increase in the abundance of the A system and the Na+,K+-ATPase is the result of a mutation in regulatory gene R1 that controls the A system and the Na+,K+-ATPase and is not due to a primary effect of a possible initial increase in Na+ concentration.

Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1.

Growth of CHO-K1, a proline auxotroph, is inhibited by amino acids that prevent proline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, alar4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, alar4-H2.1 and alar4-H3.9, isolated as resistant to 50 mM and 125 mM alanine, respectively, showed increases in Vmax of proline transport through the A system that are directly proportional to their resistance to alanine. Alar4-H3.9, as compared to alar4 and CHO-K1, has six and 29 times the Vmax of proline transport through the A system and two and five times the velocity of transport through the combined ASC and P systems, respectively, and no change in system L. No double-minute or homologous staining regions were detectable in alar4-H3.9. A-system activity of alar4-H2.1 and alar4-H3.9, when grown under nonselective conditions, was stable for 20 generations and then declined. The phenotype of alar4-H3.9 is codominant with that of alar4 and partially recessive to that of CHO-K1. Membrane vesicles prepared from alar4-H3.9 show increases mainly in A-system transport. In sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of A-system active membrane vesicles and endoplasmic reticulum, two bands of molecular weight of approximately 62-66 kd and 29 kd are present in higher concentrations in alar4-H3.9 than in CHO-K1. These results are compatible with the hypothesis that the phenotype of alar4-H3.9 is the result of gene amplification of an A-system transporter structural gene and that the two bands may represent this transporter.

Control of A-system amino acid transport by a second regulatory gene R2 in Chinese hamster ovary cells CHO-K1 and the possible connection of this gene with insulin activity.

Evidence based on a study of alanine-resistant (Alar), constitutive mutants of CHO-K1 cells and the conditions that favor stimulation of the A system of amino acid activity supports the model that the A system of amino acid transport in these cells is repressible and under negative control of regulatory gene R1. In this study, we show that mutant Alar6, when grown under conditions of repression, has an A system of amino acid transport activity similar to that of the derepressed parental cell line, CHO-K1 (wild type) and of the fully constitutive mutant in gene R1, Alar4. However, the A system of Alar6 is further derepressible. The Vmax for proline transport through this system in mutant Alar6 is four times that of the parental culture, with no significant change in Km. Analysis of hybrids produced by crossing mutant Alar6 with the parental culture and with Alar4 shows that mutant Alar6 is recessive to wild type and complements mutant Alar4. Although the amino acid transport A system of CHO-K1 is stimulated by insulin, mutant alar6 is not stimulated by insulin. These results support the hypothesis that mutant alar6 results from mutation in another regulatory gene, R2, that, in conjunction with gene R1, negatively controls the expression of a structural gene for the A-system transport. Evidence also indicates that R2 gene product is not responsive to amino acids and that insulin stimulation of the A system may result from insulin inactivation of this repressor.

Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants.

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.

Regulation of the A system of amino acid transport in Chinese hamster ovary cells, CHO-K1: the difference in specificity between the apo-repressor inactivator (apo-ri) and the transporter and the characterization of the proposed apo-ri.

When amino acids that are generally transported through the A system are added to derepressed cultures of CHO-K1 cells or to cultures that are undergoing starvation-derepression, as in the co-repressor (co-r), co-inactivator (co-i), (co-ri) assay, the A system undergoes trans-inhibition, inactivation, and repression. The effect of inactivation and repression is not related to the ability of amino acids to bind to the A system transporter but supports a model in which these amino acids act as co-r's/co-i's, and by binding to a aporepressor/inactivator (apo-ri), the product of gene R1, convert it into a repressor/inactivator (ri). For example, beta-alanine acts as a strong co-r but does not inhibit proline transport through the A system. Hydroxyproline and histidine, although poor inhibitors of proline transport, are very effective as co-ri's. Diaminobutyrate, phenylalanine, alpha-keto-glutarate, pyro-glutamate, isoleucine, and valine, compounds that inhibit A system transport, listed in decreasing order of effectiveness, are all equally poor as co-ri's. Also the Km for the transport of 2-(methylamino)isobutyric acid (MeAIB) through the A system is two times the concentration of MeAIB required to produce one-half inactivation. Amino acid effectors and mutation can modify the conversion of the apo-ri to repressor (r) and inactivator (i). The apo-ri is converted by alanine, serine, proline, and MeAIB to ri, by beta-alanine and tryptophane to r, and by hydroxyproline to r and reduced i. The full constitutive and partial constitutive mutants alar4 and alar2, respectively, are in the same complementation group. Alar4 has no active apo-ri while the rate of derepression of alar2 is twice and the inactivation rate is equal to that of the parent culture.

A defined medium for and the effect of insulin on the growth, amino acid transport, and morphology of Chinese hamster ovary cells, CHO-K1 (CCL 61) and the isolation of insulin "independent" mutants.

Insulin, FeSO4, or transferrin are major requirements together with HEPES buffer and selenium for the growth of CHO-K1 (CCL 61) in a modified F12 medium (M-F12). Insulin stimulates growth at 1 ng/ml to 10 micrograms/ml. In the defined medium minus insulin, CHO-K1 grows slowly as elongated, elliptical cells in parallel arrays typical of normal diploid fibroblasts in contrast to round-to-cuboid cells in loosely overlapping arrays in the presence of serum or insulin. During prolonged incubation in the absence of insulin the cells gather up into a large spherical cluster of viable cells. Insulin "independent" mutants have been isolated whose growth rate during exponential phase in the absence of insulin (48 h to 84 or 96 hrs) is 2.7 to 3.6 times that of the parental culture. Insulin stimulates the growth of these variants only during the first 48 h and is inhibitory at 50 to 500 ng/ml during the exponential phase. Insulin induction of the A system of amino acid transport occurs in about 8 h and requires both protein and RNA synthesis.

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.

Recessive 2-(methylamino)-isobutyrate (MeAIB)-resistant mutant of Chinese hamster ovary cells (CHO-K1) with increased transport through ASC system.

Mutants of Chinese hamster ovary cells (CHO-K1 Pro-), resistant to the proline transport antagonist 2-(methylamino)-isobutyrate (MeAIB) were isolated by a single-step procedure. Mutation rates to Pro+ and to Pro- MeAIB resistance (MeAIBr) are 1.7 X 10(-6) and 2.4 X 10(-5), respectively. Several Pro- MeAIBr mutants were tested by measuring the uptake of 0.05 mM proline through the various amino acid transport systems: some showed increases in one transport system only; others revealed pleiotropic changes affecting two or more systems; still others had no apparent change in proline transport. One Pro- MeAIBr mutant analyzed in detail (MeAIBr22) was isolated after EMS treatment as resistant to 5 mM MeAIB, is Pro-, stable, and shows a 1.6-fold increase in the initial velocity of transport of 0.05 mM proline. There appears to be no change in the velocity of proline transport through the amino acid transport systems A, P, and L, and the "glutamine inhibitable fraction." In contrast, there is a 5.5-fold increase in the velocity of transport of 0.05 mM proline through the ASC system. Kinetic studies reveal a sixfold increase in the Vm and a slight increase in the Km of the transport of serine through the ASC system. Hybrids between MeAIBr22 and CHO-K1 Pro-, OUAr, HPRT- showed the parental phenotype. These results indicate that the mutant ASC phenotype of MeAIBr22 is recessive and is probably the result of a regulatory gene mutation.

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems.

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro- alar clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by alar2 and alar3 can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The Vmax for proline transport through the A system has increased two times for alar2 while the Km and Vmax for alar3 has increased by 1.4 and 2.3 times that of CHO-K1. There is a corresponding increase in Vmax of proline transport by alar2 through the P system. The P system is defined operationally as that portion of the Na+-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li+ cannot be substituted for Na+. The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.

Inhibition of growth of proline-requiring Chinese hamster ovary cells (CHO-k1) resulting from antagonism by a system amino acids.

CHO-K1 requires proline for growth. Two proline-independent revertants were isolated--K1-J and K1-6. CHO-K1 pro- is much more sensitive than the pro+ cell lines to inhibition of growth by addition to the medium of amino acids and amino acid analogues that are transported through the A system. In contrast, pro+ cells are as sensitive as, or in some cases slightly more sensitive than, pro- cells to glycine, basic amino acids, and to amino acids that are mainly transported by the L system. The A system analogue alpha(methylamino) isobutyric acid (MAIB) in low concentrations reacts competitively with proline to regulate the growth of pro- cells, yielding a Ki for MAIB of 0.56 mM. CHO-K1 and K1-6 transport proline at the same initial rate and are equally sensitive to the inhibition of proline transport by alanine. Alanine and MAIB inhibit proline transport strongly and similarly in CHO-K1. Thus although these compounds inhibit the transport of proline by both cell types to the same extent, pro+ cells are immune to the effect of this starvation since they are able to synthesize their own proline. We also describe a secondary inhibition caused by high A system amino acid concentrations that affects both pro- and pro+ cells.

Inhibition of growth of cells in culture by L-phenylalanine as a model system for the analysis of phenylketonuria. I. aminoacid antagonism and the inhibition of protein synthesis.

Phenylalanine in high concentrations inhibits the growth of mouse A9 cells. Protein synthesis is inhibited earlier and more severely than RNA or DNA synthesis. Phenylalanine inhibits the uptake and decreases the intracellular pool of several amino acids. Certain amino acids added in excess reverse the phenylalanine inhibition. The strongest reversing amino acids appear to function by excluding phenylalanine. The phenylalanine inhibition does not appear to be due to a deficiency of any amino acid, but to the high intracellular phenylalanine concentration and/or an amino acid imbalance resulting from the large ratio of phenylalanine to other amino acids.