scholarly journals Purification and properties of peroxisomal pyruvate (glyoxylate) aminotransferase from rat liver

1978 ◽  
Vol 175 (2) ◽  
pp. 765-768 ◽  
Author(s):  
T Noguchi ◽  
Y Takada
Keyword(s):  
Amino Acids ◽  
Kinetic Parameters ◽  
Rat Liver ◽  
Isoelectric Point ◽  
Enzyme Preparation ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Amino Donor ◽  
Glyoxylate Aminotransferase ◽  
Liver Peroxisomes

Pyruvate (glyoxylate) aminotransferase from rat liver peroxisomes was highly purified and characterized. The enzyme preparation has a mol.wt. of approx. 80,000 with two identical subunits, and isoelectric point of 8.0 and a pH optimum between 8.0 and 8.5. The enzyme catalysed transamination between a number of L-amino acids and pyruvate or glyoxylate. The effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine, and glyoxylate and phenylpyruvate with alanine as amino donor. These properties and kinetic parameters of the enzyme are remarkably similar to those previously described for mitochondrial alanine-glyoxylate aminotransferase isoenzyme 1 from glucagon-injected rat liver [Noguchi, Okuno, Takada, Minatogawa, Okai & Kido (1978, Biochem. J. 169, 113-122].

Isolation and Purification to Apparent Homogeneity of 4,5-Dioxovalerate Aminotransferase from Scenedesmus obliquus Mutant C-2 A′

1988 ◽  
Vol 43 (7-8) ◽  
pp. 563-571 ◽  
Author(s):  
A. Kah ◽  
D. Dörnemann ◽  
H. Senger
Keyword(s):  
Molecular Weight ◽  
Green Alga ◽  
Isoelectric Point ◽  
Pyridoxal Phosphate ◽  
Scenedesmus Obliquus ◽  
Ph Optimum ◽  
Isolation And Purification ◽  
Apparent Homogeneity ◽  
Unicellular Green Alga ◽  
Glyoxylate Aminotransferase

In the present paper the purification of a specific 4,5-dioxovalerate transaminase from pigment mutant C-2 A′ of the unicellular green alga Scenedesmus obliquus to apparent homogeneity is described. The newly isolated enzyme ʟ-glutamate: 4,5-dioxovalerate aminotransferase is not identical with ʟ-alanine: 4,5-dioxovalerate aminotransferase (EC 2.6.1.43) and ʟ-alanine: glyoxylate aminotransferase (EC 2.6.1.44). A procedure for the purification is described and the resulting homogeneous protein is characterized by its Kᴍ-values for oxo-substrates and amino donors, its pyridoxal phosphate requirement, reversability of the catalysis, pH-optimum, isoelectric point and its molecular weight.

Glutamate Dehydrogenase from Pea Roots: Purification and Properties of the Enzyme

1971 ◽  
Vol 49 (1) ◽  
pp. 127-138 ◽  
Author(s):  
E. Pahlich ◽  
K. W. Joy
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Constant Ratio ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Activity Ratios ◽  
Single Protein ◽  
Michaelis Constants ◽  
Pea Roots

Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) has been purified 1250-fold from pea roots. The preparation contains only a single protein, and the molecular weight was estimated to be 208 000 ± 10 000. The enzyme shows NADH (aminating) and NAD+ (deaminating) activities, but the ratio of these activities is not constant and can be changed experimentally. NADPH activity is also present and shows a relatively constant ratio to NAD+ activity. EDTA inhibits NADH activity in intermediate concentrations, but reactivates at higher concentrations. NAD+ (and NADPH) activity is only slightly changed by EDTA. The effects of dioxane and the coenzymes on the enzyme are also reported. Mechanisms which could explain the different activity ratios, in terms of two interconvertible enzyme forms, are discussed.The pH optimum for NADH and NAD+ activities is about pH 8.0. Michaelis constants were found to be: α-ketoglutarate, 3.3 × 10−3 M; ammonium (sulfate), 3.8 × 10−2 M; glutamate, 7.3 × 10−3 M; NADH, 8.6 × 10−4 M; NAD+, 6.5 × 10−4 M. The enzyme is highly specific for the substrates glutamate and α-ketoglutarate, showing no alanine or aspartate dehydrogenase activity, and no deamination with a range of amino acids.

scholarly journals The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1972 ◽  
Vol 127 (1) ◽  
pp. 155-165 ◽  
Author(s):  
E. V. Rowsell ◽  
K. Snell ◽  
J. A. Carnie ◽  
Kathleen V. Rowsell
Keyword(s):  
Rat Liver ◽  
High Speed ◽  
Mitochondrial Enzymes ◽  
Aminotransferase Activity ◽  
Ph Optimum ◽  
Cytosol Fraction ◽  
Specific Association ◽  
Differential Sedimentation ◽  
Glyoxylate Aminotransferase ◽  
Glucose Formation

1. The distribution of l-alanine–glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the `total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent Km of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0–9.0) and has an apparent Km of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine–glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.

scholarly journals Characteristics of hepatic alanine-glyoxylate aminotransferase in different mammalian species

1978 ◽  
Vol 169 (1) ◽  
pp. 113-122 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Takada ◽  
Y Minatogawa ◽  
K Okai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Physical Properties ◽  
Mouse Liver ◽  
Mammalian Species ◽  
The Other ◽  
Enzyme Preparations ◽  
Amino Donor ◽  
Glyoxylate Aminotransferase

Mitochondrial extracts of dog, cat, rat and mouse liver contain two forms of alanine-glyoxylate aminotransferase (EC 2.6.1.44): one, designated isoenzyme 1, has mol.wt. approx. 80 000 and predominates in dog and cat liver; the other, designated isoenzyme 2, has mol.wt. approx. 175 000 and predominates in rat and mouse liver. In rat and mouse liver, isoenzyme 1 activity was increased by the injection in vivo of glucagon, but not isoenzyme 2 activity. Isoenzyme 1 was purified and characterized from liver mitochondrial extracts of the four species. Both rat and mouse enzyme preparations catalysed transamination between a number of L-amino acids and glyoxylate, and with L-alanine as amino donor the effective amino acceptors were glyoxylate, phenylpyruvate and hydroxypyruvate. In contrast, both dog and cat enzyme preparations were specific for L-alanine and L-serine with glyoxylate, and used glyoxylate and hydroxypyruvate as effective amino acceptors with L-alanine. Evidence that isoenzyme 1 is identical with serine-pyruvate aminotransferase (EC 2.6.1.51) was obtained. Isoenzyme 2 was partially purified from mitochondrial extracts of rat and mouse liver. Both enzyme preparations were specific for L-alanine and glyoxylate. On the basis of physical properties and substrate specificity, it was concluded that isoenzyme 2 is a separate enzyme. Some other properties of isoenzymes 1 and 2 are described.

scholarly journals Purification and properties of rat brain pyruvate carboxylase

1975 ◽  
Vol 145 (1) ◽  
pp. 25-35 ◽  
Author(s):  
D E Mahan ◽  
I K Mushahwar ◽  
R E Koeppe
Keyword(s):  
Polyethylene Glycol ◽  
Rat Brain ◽  
Kinetic Parameters ◽  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Purification And Properties

Rat brain pyruvate carboxylase was purified 2000-fold and some of its properties and kinetic parameters were investigated. The use of (NH4)2SO4 gradient solubilization on a Celite column and precipitation with polyethylene glycol permitted purification to an estimated 20% purity. Except for a few subtle kinetic differences this enzyme is indistinguishable from rat liver pyruvate carboxylase.

scholarly journals Purification and properties of the uroporphyrinogen decarboxylase from Rhodobacter sphaeroides

1993 ◽  
Vol 293 (3) ◽  
pp. 703-712 ◽  
Author(s):  
R M Jones ◽  
P M Jordan
Keyword(s):  
Amino Acids ◽  
Acetic Acid ◽  
Rhodobacter Sphaeroides ◽  
Uroporphyrinogen Decarboxylase ◽  
Ph Optimum ◽  
Purification And Properties ◽  
N Terminus ◽  
Arginine Residues ◽  
High Degree ◽  
Degree Of Similarity

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified 600-fold from Rhodobacter sphaeroides grown anaerobically in the light. The enzyme, under both denaturing and non-denaturing conditions, is a monomer of M(r) 41,000. The Km values are 1.8 microM and 6.0 microM for the conversion of uroporphyrinogen I and III to coproporphyrinogen I and III respectively. The enzyme is susceptible to inhibition by both uroporphyrinogen and uroporphyrin. The pH optimum is 6.8 and the isoelectric point is 4.4. The importance of cysteine and arginine residues is implicated from studies with inhibitors. The sequence of the first 29 amino acids of the N-terminus shows a high degree of similarity to the primary structures of other uroporphyrinogen decarboxylases. Studies on the order of decarboxylation of the four acetic acid side chains of uroporphyrinogen III suggest that at high substrate levels a random route is preferred.

Purification and properties of β-xylosidase from Penicillium wortmanni

1978 ◽  
Vol 56 (1) ◽  
pp. 43-50 ◽  
Author(s):  
F. Deleyn ◽  
M. Claeyssens ◽  
J. Van Beeumen ◽  
C. K. De Bruyne
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Ph Optimum ◽  
Purification Method ◽  
Acetone Precipitation ◽  
Purification And Properties ◽  
Tryptophan Residues ◽  
Rapid Inactivation ◽  
Inversion Mechanism

A purification method for an extracellular β-xylosidase (β-D-xyloside xylohydrolase, EC 3.2.1.37) induced in Penicillium wortmanni is described. It includes diafiltration, acetone precipitation, and hydroxylapatite chromatography. The enzyme has a molecular weight of about 100 000. Its pH optimum is at pH 3.3–4.0 and it is most stable at pH 5.0–6.0. Its isoelectric point is at pH 5.0. Sulfhydryl and histidine reagents are not inhibitory. The influence of added cations and anions is negligible. N-Bromosuccinimide oxidation of two to three tryptophan residues per molecule entails rapid inactivation. Glycon-specificity studies indicate strict requirements at C-2, C-3, C-4, and C-5, although α-L-arabinopyranosides are substrates. As the enzyme seems to hydrolyse xylooligosaccharides endwise, with retention of configuration in the reaction product, the enzyme is a true glycosidase, probably operating by a double-inversion mechanism.

scholarly journals The purification and properties of microsomal palmitoyl-coenzyme A synthetase

1971 ◽  
Vol 122 (3) ◽  
pp. 353-362 ◽  
Author(s):  
J. Bar–Tana ◽  
G. Rose ◽  
B. Shapiro
Keyword(s):  
Fatty Acid ◽  
Kinetic Parameters ◽  
Rat Liver ◽  
Enzyme Assay ◽  
Liver Microsomes ◽  
Acid Activation ◽  
Rat Liver Microsomes ◽  
Isolation And Purification ◽  
Purification And Properties ◽  
Fatty Acid Activation

The isolation and purification of palmitoyl-CoA synthetase from rat liver microsomes is described. Several methods suitable for enzyme assay are described. The general properties and kinetic parameters of the purified enzyme were determined and are discussed in relationship to microsomal fatty acid activation.

Synthetic 1-thio-β-D-glucosiduronic acids as substrates for rat-liver β-glucuronidase

1970 ◽  
Vol 48 (7) ◽  
pp. 799-804 ◽  
Author(s):  
C. Hétu ◽  
R. Gianetto
Keyword(s):  
Rat Liver ◽  
Molecular Sieve ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Filtration Step ◽  
Hydrolysis Of ◽  
Menten Constant

The hydrolysis of 1-thio-β-D-glucosiduronic acids by rat liver was studied using synthetic phenyl 1-thio-β-D-glucosiduronic acid, sodium (2-benzothiazolyl 1-thio-β- D-glucosid)uronate, and sodium (p-nitrophenyl 1-thio-β-D-glucosid)uronate. It was found that rat liver preparations can hydrolyze the β-D-glucuronides of 2-benzothiazolethiol and p-nitrothiophenol but not the β-D-glucuronide of thiophenol.Partial purification of the enzyme from a lysosomal preparation using ammonium sulfate fractionation, gel filtration on a molecular sieve, and anion-exchange chromatography showed that β-glucuronidase (EC 3.2.1.31) is the enzyme responsible for the hydrolysis of these thioglucuronides. The pH optimum and Michaelis–Menten constant (Km) were determined for both substrates using an enzyme preparation obtained after the gel filtration step. The glucuronide of 2-benzothiazolethiol was found to be almost as good a substrate as that of phenolphthalein for rat-liver β-glucuronidase, while the glucuronide of p-nitrothiophenol is hydrolyzed at a much slower rate. Possible explanations of the fact that β-glucuronidase hydrolyzes only certain thioglucuronides are suggested.

The effect of cell-sap fractions on incorporation of amino acids into protein in rat liver cell-free systems

1970 ◽  
Vol 48 (7) ◽  
pp. 735-739 ◽  
Author(s):  
C. C. Liew ◽  
A. Korner
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Rat Liver ◽  
Liver Cell ◽  
Enzyme Preparation ◽  
Transfer Factors

The ability of rat liver cell sap, pH 5 enzyme preparation, partially purified transfer factors I and II (PTF), and a mixture of the latter two to support protein synthesis in a ribosome system in vitro was compared. Protein synthesis increased as the ratio of pH 5 enzyme to ribosomes and PTF was raised and as the ratio of PTF to pH 5 enzymes and ribosomes was raised. The best ratios of these fractions were not as efficient as unfractionated cell sap at supporting protein synthesis; some factor had been lost or inactivated during fractionation. Incubation with cell sap caused changes in ribosomal profiles leading to less polysomes and more monomeric and dimeric ribosomes but no changes were noted on incubation with pH 5 enzymes and PTF.

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