The isolation and properties of phenylalanine hydroxylase from human liver

Woo, Savio L. C.; Gillam, Shirley; Woolf, L. I.

doi:10.1042/bj1390741

Cited by 50 publications

(22 citation statements)

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“…Abbreviations: cDNA = DNA complementary to an mRNA; HPA = hyperphenylalaninemia; PKU -phenylketonuria; mRNA = messenger RNA; RFLP = restriction fragment lengt polymorphism; BHy = tetrahydrobiopterin be isolated from liver tissue [17]. Antibodies against the enzyme protein will precipitate not only the enzyme but also the polysomes (Fig.…”

Section: Offprint Requests To: F Giittlermentioning

confidence: 99%

Molecular biology of phenylketonuria

et al. 1987

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Section: Offprint Requests To: F Giittlermentioning

confidence: 99%

Molecular biology of phenylketonuria

et al. 1987

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“…Phenylalanine hydroxylase has been purified from livers of rat, monkey, and human (7)(8)(9). It is a multimeric enzyme with a subunit molecular weight of about 50,000.…”

mentioning

confidence: 99%

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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“…Human phenylalanine hydroxylase has been classified as an unstable protein, which rapidly loses its enzymatic activity in post-mortem tissues or in vitro, after purification [30]. It presents a short half-life, which has been estimated to be 9.4 h in hepatoma cells and 2 days in the liver [31].…”

Section: Discussionmentioning

confidence: 99%

Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

Nascimento

Leandro

Lino

et al. 2009

Appl Biochem Biotechnol

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Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and -20 degrees C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at -20 degrees C, protected the enzyme from loss of its enzymatic activity. The determined DeltaG(0) and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.

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The isolation and properties of phenylalanine hydroxylase from human liver

Cited by 50 publications

References 9 publications

Molecular biology of phenylketonuria

Molecular biology of phenylketonuria

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

Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

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