Distribution of phenylalanine hydroxylase (EC 1.14.3.1) in liver and kidney of vertebrates

Hsieh, Monica; Berry, Helen K.

doi:10.1002/jez.1402080204

Cited by 31 publications

(19 citation statements)

References 16 publications

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“…Hybridization signals were obtained from the liver samples as expected. Phenylalanine hydroxylase activity has been reported in the kidneys of rats (30) but not primates (31). The detection of phenylalanine hydroxylase mRNA in the rat kidney but not the baboon kidney is thus completely consistent with these observations.…”

Section: Discussionsupporting

confidence: 79%

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

Robson¹,

Chandra²,

MacGillivray³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, ofwhich it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning ofits cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis ofphenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans.Phenylketonuria (PKU) is a human disorder caused by an inborn error in aromatic amino acid metabolism. Classical phenylketonuria is characterized by a complete lack of phenylalanine hydroxylase activity [phenylalanine 4-monooxygenase; L-phenylalanine,tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] in the liver (1). The enzyme normally catalyzes the oxidation of phenylalanine to tyrosine, the major metabolic pathway of phenylalanine (2). The lack of this enzymatic activity causes persistent hyperphenylalaninemia and minor metabolic pathways for phenylalanine become overutilized (3,4). High levels of phenylalanine and its derivatives are toxic and cause disturbances in tyrosine and tryptophan metabolism (5). Diminished formation of catecholamines, melanin, and serotonin is typical in untreated phenylketonuric patients, and the clinical symptom is severe and permanent mental retardation (5). The condition is autosomal recessive and has a prevalence of about 1 in 10,000 (for review, see ref. 6).Phenylalanine hydroxylase has been purified from livers of rat, monkey, and human (7-9). It is a multimeric enzyme with a subunit molecular weight of about 50,000. Recent evidence has suggested that either the subunits are identical or one is a proteolytic product of the other (10, 11). In order to gain abetter understanding ofthe molecular basis for phenylketonuria at the gene level, we have enriched phenylalanine hydroxylase mRNA from total rat liver polysomes by specific immunoprecipitation and then used the mRNA for the synthesis and cloning of its complementary DNA. METHODSPurification of Phenylalanine Hydroxylase and Its Antibody. Rat liver phenylalanine hydroxylase was purified by a modification of the procedure described by Shiman et aL (12). Enzyme activity was measured by using the fluorimetric assay for tyrosine (13), and the purity of the enzyme preparation was analyzed by NaDodSO4/polyacrylamide slab gel electrophoresis (14). Purified rat liver phenylalanine hydroxylase was used to immunize a goat, and immunospecificity ofthe antiserum was analyzed by immunodiffusion (15). Specific immunoglobulin molecules against phenylalanine hydroxylase were purified from the crude antiserum by affinity chromatography...

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Section: Discussionsupporting

confidence: 79%

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

Robson¹,

Chandra²,

MacGillivray³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Up to 40% PAH activity has been reported in the kidneys, as compared to the liver in human tissue [36]. In this study, we report 33% of kidney PAH activity in wild-type mouse tissue in relation to PAH activity in liver, in accordance with previous data in rodents [37]. In addition to liver and kidneys, significant PAH activity and expressions were also reported in pancreas tissue [36,38].…”

Section: Discussionsupporting

confidence: 90%

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Heintz

Troxler

Martı́nez

et al. 2012

Molecular Genetics and Metabolism

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BACKGROUND: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient's genotype. Activity of in vitro expressed mutant PAH may predict the patient's phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH. METHODS: A robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. RESULTS: The PAH assay was linear for phenylalanine and tyrosine (r(2) 0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 mol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. CONCLUSIONS: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions

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“…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, 0.002 M tyrosine. (Frozen extract was thawed and sonicated, 3 times for 5 s/time (0°C).)…”

Section: Quantitation Of Tyrosine and Tyrosine Degradation Products Imentioning

confidence: 99%

“…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 of DNA ϭ nmol/h/mg dry weight/600. 3 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 ϳ0.45 times the K m ) on a hyperbolic saturation curve.…”

mentioning

confidence: 96%

“…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)(6)(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.…”

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confidence: 99%

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Formation and Fate of Tyrosine

Shiman

Gray²

1998

Journal of Biological Chemistry

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Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.In animals, tyrosine is formed by hydroxylation of phenylalanine in a reaction catalyzed by phenylalanine hydroxylase (1, 2). The reaction occurs almost exclusively in liver (3), it is the first step in phenylalanine degradation (1, 4), and phenylalanine appears to be its primary regulator (5-7). Since liver is the site of tyrosine degradation as well as formation, the size of the intracellular tyrosine pool and the relative rates of tyrosine synthesis and degradation might be expected to depend, at least in part, on tyrosine concentration. Tyrosine could directly or indirectly (e.g. through the phenylalanine hydroxylase cofactor tetrahydrobiopterin (1,8)) affect the rate of phenylalanine hydroxylation, it could affect the rate of tyrosine degradation, or it could affect the disposition (metabolic routing) of tyrosine in the cell. The mechanisms are not mutually exclusive, and all could operate, although a direct effect of tyrosine on phenylalanine hydroxylase has not, so far, been observed either in studies with purified enzyme or in the few in situ experiments that have addressed this question (9).Historically, studies of regulation of the tyrosine pool have focused on tyrosine degradation, and most of the interest in degradation has been on control of tyrosine aminotransferase which catalyzes the first step in the pathway, deamination to p-hydroxyphenylpyruvate. The tyrosine aminotransferase reaction, although reversible, is usually considered the rate-limiting and regulated step in tyrosine metabolism (see Refs. 10 -12, but see also ...

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Distribution of phenylalanine hydroxylase (EC 1.14.3.1) in liver and kidney of vertebrates

Cited by 31 publications

References 16 publications

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry

Formation and Fate of Tyrosine

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