The isolation and properties of phenylalanine hydroxylase from human liver

Savio L. C. Woo; Shirley Su Gillam; Louis I. Woolf

doi:10.1042/bj1390741

The isolation and properties of phenylalanine hydroxylase from human liver

Biochemical Journal ◽

10.1042/bj1390741 ◽

1974 ◽

Vol 139 (3) ◽

pp. 741-749 ◽

Cited By ~ 42

Author(s):

Savio L. C. Woo ◽

Shirley Su Gillam ◽

Louis I. Woolf

Keyword(s):

Phenylalanine Hydroxylase ◽

Term Infant ◽

Molecular Weights ◽

Human Phenylalanine Hydroxylase ◽

Normal Enzyme ◽

High Concentrations ◽

Thiol Binding ◽

Filtration Step ◽

Preliminary Filtration ◽

Human Foetal Liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The Km values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.

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The isolation and properties of phenylalanine hydroxylase from rat liver

Biochemical Journal ◽

10.1042/bj1390731 ◽

1974 ◽

Vol 139 (3) ◽

pp. 731-739 ◽

Cited By ~ 22

Author(s):

Shirley Su Gillam ◽

Savio L. C. Woo ◽

Louis I. Woolf

Keyword(s):

Rat Liver ◽

Phenylalanine Hydroxylase ◽

Protein Band ◽

Enzymic Activity ◽

Hydroxylation Reaction ◽

Single Protein ◽

Thiol Binding ◽

Filtration Step ◽

Preliminary Filtration ◽

Haem Iron

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The Km and Vmax. values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.

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Orphan designation: Adeno-associated virus serotype HSC15 expressing human phenylalanine hydroxylase, Treatment of phenylalanine hydroxylase deficiency

Case Medical Research ◽

10.31525/cmr-d17e13 ◽

2019 ◽

Author(s):

Keyword(s):

Phenylalanine Hydroxylase ◽

Hydroxylase Deficiency ◽

Adeno Associated Virus ◽

Orphan Designation ◽

Virus Serotype ◽

Human Phenylalanine Hydroxylase

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Calcium-sensitive, lipid-binding cytoskeletal proteins of the human placental microvillar region.

The Journal of Cell Biology ◽

10.1083/jcb.105.1.303 ◽

1987 ◽

Vol 105 (1) ◽

pp. 303-311 ◽

Cited By ~ 29

Author(s):

H C Edwards ◽

A G Booth

Keyword(s):

Lipid Binding ◽

Cytoskeletal Proteins ◽

Dependent Manner ◽

Molecular Weights ◽

Sequence Of Events ◽

Marked Preference ◽

High Concentrations

In this study we describe a group of Ca2+-sensitive proteins located in the microvillar region of the human placental syncytiotrophoblast. By following the distribution of proteins between the particulate and supernatant phases of detergent-solubilized microvilli in the presence of defined concentrations of free Ca2+, we demonstrate a class of proteins of subunit molecular weights 72,000, 69,000, 38,000, 36,000, and 32,000 that associate with both the cytoskeleton and lipid at high concentrations of free Ca2+. These proteins can be released from microvilli using EGTA-containing buffers. Although they do not bind to phenyl-Sepharose, they will bind to phospholipids immobilized on phenyl-Sepharose columns in a Ca2+-dependent manner and show a marked preference for phospholipids with negatively charged headgroups. The results provide evidence for a sequence of events which may occur within the microvillus as the localized concentration of intracellular free Ca2+ rises.

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Structural characterization of the 5' regions of the human phenylalanine hydroxylase gene

Biochemistry ◽

10.1021/bi00150a033 ◽

1992 ◽

Vol 31 (35) ◽

pp. 8363-8368 ◽

Cited By ~ 28

Author(s):

David S. Konecki ◽

Yibin Wang ◽

Friedrich K. Trefz ◽

Uta Lichter-Konecki ◽

Savio L. C. Woo

Keyword(s):

Structural Characterization ◽

Phenylalanine Hydroxylase ◽

Hydroxylase Gene ◽

Phenylalanine Hydroxylase Gene ◽

Human Phenylalanine Hydroxylase

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Proteolytic and peroxidatic reactions of commercial horseradish peroxidase with myelin basic protein

Biochemical Journal ◽

10.1042/bj1690567 ◽

1978 ◽

Vol 169 (3) ◽

pp. 567-575 ◽

Cited By ~ 9

Author(s):

Wendy Cammer ◽

Lesley Z. Bieler ◽

William T. Norton

Keyword(s):

Horseradish Peroxidase ◽

Myelin Basic Protein ◽

Basic Protein ◽

Protein A ◽

Low Molecular Weight ◽

Basic Proteins ◽

Molecular Weights ◽

Low Concentrations ◽

High Concentrations ◽

A Minor

Degradation of myelin basic protein during incubations with high concentrations of horseradish peroxidase has been demonstrated [Johnson & Cammer (1977) J. Histochem. Cytochem.25, 329–336]. Possible mechanisms for the interaction of the basic protein with peroxidase were investigated in the present study. Because the peroxidase samples previously observed to degrade basic protein were mixtures of isoenzymes, commercial preparations of the separated isoenzymes were tested, and all three degraded basic protein, but to various extents. Three other basic proteins, P2 protein from peripheral nerve myelin, lysozyme and cytochrome c, were not degraded by horseradish peroxidase under the same conditions. Inhibitor studies suggested a minor peroxidatic component in the reaction. Therefore the peroxidatic reaction with basic protein was studied by using low concentrations of peroxidase along with H2O2. Horseradish peroxidase plus H2O2 caused the destruction of basic protein, a reaction inhibited by cyanide, azide, ferrocyanide, tyrosine, di-iodotyrosine and catalase. Lactoperoxidase plus H2O2 and myoglobin plus H2O2 were also effective in destroying the myelin basic protein. Low concentrations of horseradish peroxidase plus H2O2 were not active against other basic proteins, but did destroy casein and fibrinogen. Although high concentrations of peroxidase alone degraded basic protein to low-molecular-weight products, suggesting the operation of a proteolytic enzyme contaminant in the absence of H2O2, incubations with catalytic concentrations of peroxidase in the presence of H2O2 converted basic protein into products with high molecular weights. Our data suggest a mechanism for the latter, peroxidatic, reaction where polymers would form by linking the tyrosine side chains in basic-protein molecules. These data show that the myelin basic protein is unusually susceptible to peroxidatic reactions.

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Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties

Journal of Cell Science ◽

10.1242/jcs.93.1.123 ◽

1989 ◽

Vol 93 (1) ◽

pp. 123-131

Author(s):

NANCY J. LANE ◽

STEPHEN M. DILWORTH

Keyword(s):

Periplaneta Americana ◽

Biochemical Characterization ◽

Septate Junction ◽

Septate Junctions ◽

Molecular Weights ◽

Thin Sectioning ◽

Sds Page ◽

High Concentrations ◽

Membrane Components

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.

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Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Blood ◽

10.1182/blood.v64.5.1094.1094 ◽

1984 ◽

Vol 64 (5) ◽

pp. 1094-1102 ◽

Cited By ~ 2

Author(s):

Y Ozaki ◽

J Iwata ◽

T Ohashi

Keyword(s):

Membrane Proteins ◽

Wheat Germ Agglutinin ◽

Wheat Germ ◽

Sodium Dodecyl ◽

Submaxillary Gland ◽

Binding Property ◽

Molecular Weights ◽

Acetylneuraminic Acid ◽

Cell Extracts ◽

High Concentrations

Abstract Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

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LIPOPROTEIN BINDING TOHUMAN PLATELETS IS LOCATED AT GPIIb/IIIa COMPLEX

10.1055/s-0038-1643702 ◽

1987 ◽

Author(s):

E Koller ◽

F Koller

Keyword(s):

Specific Binding ◽

Human Platelets ◽

Plasma Lipoproteins ◽

High Density Lipoproteins ◽

Molecular Weights ◽

Disulfide Reduction ◽

Specific Binding Sites ◽

First Results ◽

Lipoprotein Binding ◽

High Concentrations

Human Platelets possess specific binding sites for low density lipoproteins (LDL) and high density lipoproteins(HDL)(1). Binding of both classes of plasma lipoproteins, though competitive, has been shown by several groups to facilitate platelet activation.Isolated washed platelets occasionally aggregate upon addition of high concentrations of LDL even in the absence of known platelet activators. The proteins responsible for this binding have been visualized by ligand blotting (2). Both types of ligand specifically bind to two glycoproteins with molecular weights of 135 and 115 kD, respectively. The conditions of binding to these two proteins, however, markedly differ from those known for other lipoprotein receptors.Following extensive purification, these two species are still present at concentrations relative to each other that depend markedly on the conditions of purification. The purified, solubilized receptor was tested under various conditions, including in the absence and presence of calcium, after disulfide-reduction, and following chymotrypsin digestion. In parallel experiments, the same preparations were tested with respect to binding of fibrinogen, different lectins, and thealloantibody anti-PlAI . The results strongly support the assumption, that the two protein bands associated with lipoprotein binding are constituents of the GP-IIb/IIIa complex.These first results may have greatimplications for our understanding ofthe mechanism by which lipoproteins facilitate platelet stimulation.

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