Liver Phenylalanine Hydroxylase Research Articles

Relationship between the multiple forms of rat hepatic phenylalanine hydroxylase and degree of phosphorylation.

The two major forms of rat liver phenylalanine hydroxylase have been resolved by calcium phosphate chromatography and have been highly purified. These different forms of the enzyme contain dissimilar amounts of endogenous protein-bound phosphate. The hydroxylase activity of these forms, as assayed in the presence of tetrahydrobiopterin, can be differentially stimulated by treatment with Mg2+, ATP, cyclic AMP, and protein kinase, leading to the production of a chromatographically distinct species. In the presence of [gamma-32P]ATP, activation is accompanied by differential incorporation of 32P into the subunits (Mr = 50,000) of the two enzyme forms leading to a new form which contains 1 mol of phosphate/subunit. In addition, phosphorylation in vitro eliminates the electrophoretic heterogeneity displayed by the isolated forms. These data show that the multiple forms and electrophoretic heterogeneity of phenylalanine hydroxylase are correlated with the degree of phosphorylation and also provide further evidence for the regulation of this enzyme by phosphorylation and dephosphorylation.

Amino acid sequence at the phosphorylated site of rat liver phenylalanine hydroxylase and phosphorylation of a corresponding synthetic peptide

Purified rat liver phenylalanine hydroxylase was phosphorylated by incubation with ( 32P)ATP and the catalytic subunit of pig muscle cyclic AMP-stimulated protein kinase. After digestion of the phosphorylated phenylalanine hydroxylase with pepsin, one major ( 32P)phosphopeptide with the amino acid sequence Ser-Arg-Lys-Leu-( 32P)SerP-Asx-Phe-Gly-Glx-Glx was isolated. Two non-phosphorylated octapeptides, Ser-Arg-Lys-Leu-Ser-Asp-Phe-Gly and Ser-Arg-Lys-Leu-Ser-Asn-Phe-Gly, were synthesized and characterized as substrates of the protein kinase.

Observations indicating the nature of the mutation in phenylketonuria.

Rabbit antiserum was prepared against purified normal human liver phenylalanine hydroxylase. This was used to test for cross-reacting material in crude extracts of livers from patients with classical phenylketonuria (PKU); the samples from patients were two livers obtained at autopsy and a needle biopsy core. None of these enzymically inactive livers contained detectable cross-reacting material capable of neutralizing antibody activity in double immunodiffusion and/or enzyme inhibition experiments. In an earlier study, no phenylalanine hydroxylase was found in the two autopsy PKU livers by an affinity chromatography method or by the use of a specific antiserum raised against purified monkey liver phenylalanine hydroxylase (Choo et al., 1979a). This evidence suggests that mutations in th PKU patients studied may be regulatory in nature.

Iron deficiency in the rat: effects on phenylalanine metabolism.

Concentrations of phenylalanine in the plasma were markedly elevated in iron-deficient rats and appeared to vary directly with the degree of iron deficiency. Plasma concentrations of phenylalanine returned to control levels within one week after treatment of the iron-deficient rats with iron dextran. The elevated levels of plasma phenylalanine were probably not produced by a deficiency in liver phenylalanine hydroxylase because levels of activity of the enzyme were found to be normal in the livers of the iron-deficient animals.

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.

Developmental aspect of phenylalanine hydroxylase in the rat — hormonal influences

Interpretations of the development of phenylalanine hydroxylase (PAH) in rat liver have been controversial, and the mechanism of ontogenic changes have not yet been elucidated. Fetal PAH activity at a gestational age of 21 days appeared to reach 32% that of adult male level at birth. The in vivo effectiveness of fetal PAH activity was correlated with enhancement of blood tyrosine, while tyrosine amino-transferase activity appeared only after birth. No sex difference was noted in weaning rats, whereas, in adult females, PAH activity was only 42% that of males. Investigating hormonal influences on liver PAH activity we noted no change of enzyme activity following hydrocortisone, ACTH and estradiol treatment. However, 4 days of testosterone treatment in weaning female rats increased PAH activity (X1.7). Therefore, testosterone could explain increased PAH activity in adult males.

The mechanism of the irreversible inhibition ofrat liver phenylalanine hydroxylase due to treatment with p-chlorophenylalanine. The lack of effect on turnover of phenylalanine hydroxylase.

The administration of a single dose of p-chlorophenylalanine (360 mg/kg) to rats leads to the irreversible loss of 90% of hepatic phenylalanine hydroxylase activity after 24 h. This loss of activity is not the result of either an alteration in the overall structure of the enzyme, as determined by its antigenicity, or in the total immunologically reactive protein in the liver, as tested with a specific antiserum prepared against native phenylalanine hydroxylase. Neither the rate of synthesis nor the rate of degradation of phenylalanine hydroxylase is changed by p-chlorophenylalanine (pClPhe) treatment. The half-life for the enzyme is about 2 days in control and in pClPhe-treated rats. In addition, there is no detectable incorporation of pClPhe into the phenylalanine hydroxylase molecule itself.

Beta-2-Thienyl-DL-alanine as an inhibitor of phenylalanine hydroxylase and phenylalanine intestinal transport.

The inhibitory properties of beta-2-thienyl-dl-alanine on rat phenylalanine hydroxylase from crude liver and kidney homogenates were assessed in vitro and in vivo, as well as its effects on the intestinal transport of phenylalanine, by using a perfusion procedure in vivo. The apparent K(m) for liver phenylalanine hydroxylase changed from 0.61mm in the absence of the inhibitor to 2.70mm in the presence of 24mm-beta-2-thienyl-dl-alanine, with no significant change in the V(max.). For kidney the corresponding values were 0.50 and 1.60mm respectively. A single dose of beta-2-thienyl-dl-alanine (2mmol/kg) failed to inhibit phenylalanine hydroxylase in either organ. Repeated injections during a 4-day period caused a decline of the enzymic activity to about 40% of controls. Intestinal absorption of phenylalanine when perfused at 0.2-2.0mm concentration was also competitively inhibited by beta-2-thienyl-dl-alanine. Its K(i) value was estimated at 81mm. The limited inhibitory effects of beta-2-thienyl-dl-alanine towards hepatic phenylalanine hydroxylase and phenylalanine intestinal transport, and its rapid metabolism, as suggested by the small elimination of this compound in the urine and its virtual absence from animal tissues, are factors that restrict its potential usefulness as an inducer of phenylketonuria in rats or as an effective blocker of phenylalanine absorption by the gut.

Covalent coupling of rat liver phenylalanine hydroxylase

Optimal conditions have been determined for the coupling of rat liver phenylalanine hydroxylase (Phe H) to cyanogen bromide-Sepharose 4B. When 8 mg of ligand was reacted with 100 mg of matrix, 20 to 30 percent of the initial enzyme activity was covalently bound along with 90 percent of the protein. The coupled enzyme showed greater thermal stability from 40° to 60°, a broader base of optimal pH activity from 5.8 to 10.0, more resistance to proteolysis and less inhibition by various inhibitors. The uncoupled enzyme exhibited greater storage stability at 25° after 24 hr and at 0° after 18 days. Alteration of the microenvironment by introduction of sulfhydryl groups or of carriers having positive or negative charges had variable effects on the hydroxylase activity.

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two K m values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single K m value and showed the classical Michaelis-Menten kinetics.

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Entrapment of phenylalanine hydroxylase in a polyacrylamide matrix

Rat liver phenylalanine hydroxylase has been shown to be competitively inhibited by p-chlorophenylalanine. More than 90% inhibition was produced by Ag +, Cu +, Cu 2+, and Hg 2+. It is assumed that these metal ions do not exert their action via sulfhydryl groups since iodoacetate is without effect. The observed inhibition might be due to competition with the enzyme's iron atoms by interfering with electron transport or to inactivation of the pteridine cofactor. Inhibition of hydroxylation by ammonium persulfate was caused by oxidation of the cofactor, 2-amino-4-hydroxy-6, 7-dimethyl-5, 6, 7, 8-tetrahydropteridine; no inhibition occurred in the presence of an excess of dithiothreitol. Approximately 35% of the hydroxylating activity was retained in a polyacrylamide matrix after photocatalytic polymerization. The progressive loss of phenylalanine hydroxylase activity with increasing concentrations of acrylamide was attributed to denaturation.

Partial purification and characterization of rat liver phenylalanine hydroxylase phosphatase.

A phosphatase which catalyzes the release of 32Pi from 32P-labeled phenylalanine hydroxylase has been purified about 50-fold from rat liver extracts. The phosphatase is able to catalyze the removal of only 70 to 80% of the 32Pi leaving about the same amount of Pi still bound to the hydroxylase as was originally present in the native enzyme. Dephosphorylation is accompanied by a corresponding loss in phenylalanine hydroxylase activity when the activity is measured with the natural cofactor, tetrahydrobiopterin, but not when measured with the synthetic cofactor, 6-methyltetrahydropterin. The partially purified phosphatase has very low activity toward p-nitrophenylphosphate, histone phosphate, and phosphorylase a. The activity toward these substrates has not been purified to the same extent as the phenylalanine hydroxylase phosphatase activity.

Modification of the multiple forms of rat hepatic phenylalanine hydroxylase by in vitro phosphorylation

The two major forms of rat liver phenylalanine hydroxylase have been isolated and partially purified. The tetrahydrobiopterin-dependent activity of these forms can be differentially stimulated by exposure to enzymatic phosphorylating conditions. This in vitro treatment is associated with incorporation of 32p into the enzymes and generates a further, chromatographically distinct, species. These results suggest that the multiple forms of rat liver phenylalanine hydroxylase are due to different degrees of phosphorylation.

Relation between Liver Amino Acid-Catabolizing Enzymes and Free Amino Acids of Liver and Plasma in Adult Cockerels Fed Diets Containing Graded Levels of Protein

Activities of liver amino acid-catabolizing enzymes and liver and plasma free amino acid concentrations in adult cockerels fed diets containing 3% to 21% casein were determined 2 hours after a meal. Liver tryptophan pyrrolase activity and liver tryptophan increased with an increase in dietary casein level, but plasma tryptophan decreased with the increase dietary casein level. As the dietary casein level increased, liver lysine-ketoglutarate reductase activity decreased, but liver and plasma lysine increased proportionally, and the rate of increase was larger in the plasma than in the liver. Liver phenylalanine hydroxylase activity increased with the increase in dietary casein level from 3% to 12%, and thereafter remained unchanged. Reflecting the change of this enzyme, phenylalanine increased in the liver and decreased in the plasma with the increase in dietary casein level from 3% to 12%, and thereafter the rate of increase in liver phenylalanine became small and plasms phenylalanine increased. Liver tyrosine was not influenced by the dietary casein level, whereas plasma tyrosine increased sharply from 3% to 12%, and thereafter the rate of increase decreased. Thus, the difference between liver and plasma free amino acid responses might be due to the changes in the activities of liver amino acid-catabolizing enzymes.

Development of liver phenylalanine hydroxylase and brain aromatic hydroxylases in human fetuses

Liver phenylalanine hydroxylase activity in human fetuses obtained from therapeutic abortions was found to decrease with gestational age and weight up to the second trimester of intrauterine development. After 22 weeks of gestation, enzyme levels were almost negligible. Brain tyrosine hydroxylase in fetuses 10 to 30 weeks of gestational age was very low or undetectable. There was no correlation between brain phenylalanine and tyrosine hydroxylases in the specimens analyzed.

The use of dithiothreitol in the assay of phenylalanine hydroxylase

An assay for liver phenylalanine hydroxylase based on the nonenzymatic regeneration of 6,7-dimethyltetrahydropterin from 6,7-dimethyldihydropterin (quinonoid form) by dithiothreitol is described. Dithiothreitol not only regenerates the tetrahydropteridine from dyhydropteridine but it also effectively protects the system from peroxide formed during the aerobic oxidation of the tetrahydropteridine.

Phenylalanine Hydroxylase and Tyrosine Aminotransferase in Human Fetal and Adult Liver

The mean value of phenylalanine hydroxylase activity in adult human liver was almost twice as high as that in the fetal ones; no consistent variations were seen with sex, fetal age (between the 11th and 22nd week of gestation), or with hours between delivery and death. The tyrosine aminotransferase levels did not correlate with sex, age, or method of abortion; however, they were 5 times higher in the two fetuses which survived for more than 1.5 hr after delivery. The mean concentration of tyrosine aminotransferase (excluding these two fetuses) was about 15 times lower than in the adult liver. Phenylalanine hydroxylase is known to appear in rat liver on the 20th to 21st day of gestation. Tyrosine aminotransferase, on the other hand, emerges on the first postnatal day and, as shown here, can be evoked by premature delivery. Thus, in man as in the rat, (1) phenylalanine hydroxylase approaches physiologically significant levels at an earlier development stage than does tyrosine aminotransferase and (2) extrauterinization stimulates the synthesis of tyrosine aminotransferase but not of phenylalanine hydroxylase. Speculation There is a greater analogy than is usually assumed between human and rat liver with respect to the time schedule of enzymic differentiation and the factors which regulate it.

Free amino acid pool in the brain of mice homozygous for the gene "dilute lethal".

The neurologic mutant "dilute lethal" (dl) mice, which reveal several neurologic and biochemical disturbances similar to human phenylketonuria, were used to investigate some aspects of amino acid disorder. We have studied the free amino pool in the brain of "dl" mice and of their control littermates as well as phenylalanine and tyrosine levels in brain and liver as a function of age and after phenylalamine overload. The tyrosine level decreased in brain and liver of affected mice whereas the phenylalanine/tyrosine ratio increased as a function of age. The significantly higher phenylalanine level and phenylalanine/tyrosine ratio in the liver of 20-day-old "dl" mice suggest a lower liver phenylalanine hydroxylase activity. After phenylalanine overload, the impairment of phenylalanine metabolism is predominant in the brain of "dl" mice, suggesting a disturbance in phenylalanine hydroxylation. A decrease in the level of several amino acids occurs in the brains of "dl" mice without or after phenylalanine overload; these facts might correspond to a disturbance in the transfer of amino acids to the brain and may lead to impairment in protein synthesis.

Liver phenylalanine hydroxylase assay

The first reaction is catalyzed by phenylalanine hydroxylase and the second reaction, which generates the reduced form of pteridine cofactor (biopterin), is catalyzed by dihydropteridine reductase (l-3). A direct assay of phenylalanine hydroxylase can be achieved by supplying optimal concentrations of reduced pteridine cofactor or an analog of the reduced cofactor (6,7-dimethyl-5,6,7,8-tetrahydropterine) maintained in its reduced state by the addition of NADH or NADPH (4). The assay avoids the need for an accessory NADPH enzyme-generating system and ensures that the only rate-determining component in the hydroxylating reaction is the concentration of phenylalanine hydroxylase. A typical assay for phenylalanine hydroxylase is given in Table 1. The control flasks contain all of the reagents except L-phenylalanine. Incubations are carried out at 37” in a 1Oml Erlenmeyer flask in a Dubnoff shaker, in air, and the reaction is started by