scholarly journals The isolation and properties of phenylalanine hydroxylase from rat liver

1974 ◽  
Vol 139 (3) ◽  
pp. 731-739 ◽  
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.

scholarly journals The isolation and properties of phenylalanine hydroxylase from human liver

1974 ◽  
Vol 139 (3) ◽  
pp. 741-749 ◽  
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.

Comparison of Different Methods for the Determination of Phenylalanine Hydroxylase Activity in Rat Liver and Euglena gracilis

1984 ◽  
Vol 39 (7-8) ◽  
pp. 728-733 ◽  
Author(s):  
Rita M. Fink ◽  
Erich F. Elstner
Keyword(s):  
Rat Liver ◽  
Euglena Gracilis ◽  
Phenylalanine Hydroxylase ◽  
Hplc Method ◽  
Enzymic Activity ◽  
Hydroxylase Activity ◽  
Product Separation ◽  
Tyrosine Concentration ◽  
Layer Chromatography

Abstract Three different methods for the determination of phenylalanine hydroxylase activity have been compared: a) Differential photometric assay of the increase in tyrosine concentration in the presence of phenylalanine; b) Product separation by thin layer chromatography and scintillation counting of the [14C]tyrosine formed;c) HPLC separation and spectrofluorometric quantification of derivatized amino acids. A comparison of the activities of phenylalanine hydroxylase in rat liver and Euglena gracilis clearly showed that only rat liver contains this enzymic activity as shown by methods b) and c) although pseudo-activity of Euglena gracilis preparations was found during the spectrophotometric test a). The HPLC method proved to be the fastest, most reliable and convenient method for direct tyrosine determination and thus for measuring phenylalanine hydroxylase activity.

Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

1976 ◽  
Vol 54 (5) ◽  
pp. 423-431 ◽  
Author(s):  
Kun-Tsan Lin ◽  
John C. Crawhall
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Protein Band ◽  
Final Concentration ◽  
Assay Method ◽  
Enzyme Extract ◽  
Crude Enzyme Extract ◽  
Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

scholarly journals Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3α-hydroxysteroid dehydrogenase

1994 ◽  
Vol 304 (2) ◽  
pp. 385-390 ◽  
Author(s):  
B Del Bello ◽  
E Maellaro ◽  
L Sugherini ◽  
A Santucci ◽  
M Comporti ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Dehydroascorbic Acid ◽  
Hydroxysteroid Dehydrogenase ◽  
Protein Band ◽  
Sds Page ◽  
Single Protein ◽  
Ammonium Sulphate Fractionation ◽  
Inflammatory Drugs ◽  
Rat Liver Cytosol

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

scholarly journals The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme

1987 ◽  
Vol 248 (1) ◽  
pp. 277-279 ◽  
Author(s):  
H Plat ◽  
B E Krenn ◽  
R Wever
Keyword(s):  
High Temperatures ◽  
Thermal Denaturation ◽  
Protein Band ◽  
Enzymic Activity ◽  
Single Protein Band ◽  
Xanthoria Parietina ◽  
Single Protein

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.

scholarly journals Purification and photoaffinity labelling of lipid methyltransferase from rat liver

1984 ◽  
Vol 223 (1) ◽  
pp. 61-66 ◽  
Author(s):  
M A Pajares ◽  
S Alemany ◽  
I Varela ◽  
D Marin Cao ◽  
J M Mato
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Rat Liver ◽  
Short Wavelength ◽  
Protein Band ◽  
Catalytic Subunit ◽  
Photoaffinity Labelling ◽  
Methyl Donor ◽  
Single Protein Band ◽  
Single Protein

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

scholarly journals Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column

1975 ◽  
Vol 147 (1) ◽  
pp. 131-137 ◽  
Author(s):  
M Gorecki ◽  
A Bar-Eli ◽  
Y Burstein ◽  
A Patchornik ◽  
E B Chain
Keyword(s):  
Escherichia Coli ◽  
Protein Band ◽  
Enzymic Activity ◽  
Coli Strain ◽  
Sepharose Column ◽  
Absolute Requirement ◽  
Single Protein ◽  
One Step ◽  
Triton X ◽  
Escherichia Coli B

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

scholarly journals The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver

1989 ◽  
Vol 264 (1) ◽  
pp. 185-190 ◽  
Author(s):  
D S Taylor ◽  
H H M Dahl ◽  
J F B Mercer ◽  
A K Green ◽  
M J Fisher
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Phenylalanine Hydroxylase ◽  
Enzymic Activity ◽  
Hormonal Control ◽  
Significant Elevation ◽  
Translational Activity ◽  
Streptozotocin Induced Diabetes ◽  
The Impact ◽  
Specific Mrna

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.

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