scholarly journals Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase

1976 ◽  
Vol 159 (3) ◽  
pp. 607-613 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
R Kido
Keyword(s):  
Amino Acids ◽  
Pyridoxal Phosphate ◽  
Competitive Inhibitor ◽  
Mitochondrial Fraction ◽  
Constant Ratio ◽  
Absorbance Maximum ◽  
Glucagon Injection

After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate.

scholarly journals Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ

1997 ◽  
Vol 323 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Jesús MATEO ◽  
Pedro ROTLLAN ◽  
Eulalia MARTI ◽  
Inmaculada GOMEZ DE ARANDA ◽  
Carles SOLSONA ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Pyridoxal Phosphate ◽  
Electric Organ ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Ic50 Value ◽  
Neural Origin ◽  
Disulphonic Acid ◽  
Torpedo Electric Organ ◽  
Enzyme Activators

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1,N6-ethenoadenosine) 5´,5‴-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (Apn A, where n = 3–5), producing AMP and the corresponding adenosine (n-1) 5´-phosphate, Ap(n-1). The Km values of the enzyme were 0.543± 0.015, 0.478±0.043 and 0.520±0.026 μM, and the Vmax values were 633±4, 592±18 and 576±45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5´,5‴-P1,P3-triphosphate), Ap4A (adenosine 5´,5‴-P1,P4 -tetraphosphate) and Ap5A (adenosine 5´,5‴-P1,P5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86±0.11, 1.35±0.24 and 0.58±0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38±0.19 mM. The ATP analogues adenosine 5´-tetraphosphate and adenosine 5´-[γ-thio]triphosphate are potent competitive inhibitors and adenosine 5´-[α,β-methylene]diphosphate is a less potent competitive inhibitor, the Ki values being 0.29±0.03, 0.43±0.05 and 7.18±0.8 μM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2´,4´-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7±3.1 μM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.

scholarly journals Localization and interconversion of tetrahydropteroylglutamates in isolated pea mitochondria

1972 ◽  
Vol 128 (1) ◽  
pp. 29-40 ◽  
Author(s):  
M. T. Clandinin ◽  
E. A. Cossins
Keyword(s):  
Amino Acids ◽  
Free Amino ◽  
Lactobacillus Casei ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
C1 Metabolism ◽  
Isolated Mitochondria ◽  
Feeding Experiments ◽  
Pteroylglutamic Acid ◽  
Derivatives Of

1. Mitochondria were extracted from 4-day-old pea cotyledons and purified on a sucrose density gradient. 2. Microbiological assay of the purified mitochondrial fraction with Lactobacillus casei (A.T.C.C. 7469), Streptococcus faecalis (A.T.C.C. 8043) and Pediococcus cerevisiae (A.T.C.C. 8081) revealed a discrete pool of conjugated and unconjugated derivatives of tetrahydropteroylglutamic acid. 3. Solubilization and chromatographic studies of the mitochondrial fraction demonstrated the presence of formylated and methylated derivatives, 10-formyltetrahydropteroylmonoglutamic acid, 5-formyltetrahydropteroylmonoglutamic acid and 5-formyltetrahydropteroyldiglutamic acid being the major derivatives present. 4. The principal mitochondrial pteroylglutamates were labelled when dry seeds were allowed to imbibe [2-14C]pteroylglutamic acid and 5-[methyl-14C]-methyltetrahydropteroylmonoglutamic acid. 5. The ability of isolated mitochondria to catalyse oxidation and reduction of tetrahydropteroylglutamic acid derivatives was demonstrated in feeding experiments in which [14C]formaldehyde, [3-14C]serine, sodium [14C]formate, 5-[methyl-14C]methyltetrahydropteroylmonoglutamic acid or [2-14C]-glycine served as C1 donor. In addition,14C was incorporated into free amino acids related to C1 metabolism.

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 Протеїновий і амінокислотний обмін у м’язах курчат-бройлерів кросу КОББ 500 на тлі застосування кормової добавки «Гумілід»

2017 ◽  
Vol 19 (77) ◽  
pp. 110-116
Author(s):  
E.O. Myhaylenko ◽  
O.O. Dyomshyna ◽  
L.M. Stepchenko
Keyword(s):  
Amino Acids ◽  
Broiler Chickens ◽  
Protein Biosynthesis ◽  
Mitochondrial Fraction ◽  
Feed Additives ◽  
Protective Mechanism ◽  
Mitochondrial Fractions ◽  
Adaptive Processes ◽  
Simultaneous Decrease ◽  
The Impact

The article presents data on the study of the impact of feed additives «Humilid» indicators on protein and amino acid metabolism of muscles of broiler chickens cross the COBB 500.The study tested that birds which additived Humilid the water increase in the muscles of total protein, which represented the largest share of the cytosolic and mitochondrial fractions. In homogenate of muscle, the total amount of protein increased by 10% in cytosolic and 20% in mitochondrial, which makes it possible to assert that stimulate the synthesis of cytosolic proteins is influenced Humilid and stimulated the formation chondriome of myocytes. Also, the data indicate an intensification of the use of amino acids for protein biosynthesis and adaptive processes, confirmed by increased in muscle mitochondrial fraction 2 times activity of gamma-glutamyltranspeptidase, which is involved in the transport of amino acids and glutathione in mitochondria that seen as a protective mechanism. The research has shown increased 3 times in cytosolic fraction activity of alanine aminotransferase and the simultaneous decrease in lactate dehydrogenase. Calculate the ratio activity of LDH/ALT showed bias towards anaerobic conversion of glucose to glucose-alanine cycle, more efficient way of recovery and using of glucose.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

2020 ◽  
Author(s):  
Mengbin Chen ◽  
Chun-Ting Liu ◽  
Yi Tang
Keyword(s):  
Amino Acids ◽  
Biosynthetic Pathway ◽  
Pyridoxal Phosphate ◽  
Biochemical Characterization ◽  
Indole Alkaloid ◽  
Building Blocks ◽  
Nucleophilic Attack ◽  
Keto Esters ◽  
Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2<i>S, </i>6<i>S</i>)-6-methyl pipecolate <b>1</b>, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (<i>S</i>)-2-amino-6-oxoheptanoate <b>3</b> that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives <b>1</b>. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of <i>O</i>-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C<sub>gamma</sub>-C<sub>delta</sub> bond in <b>3 </b>and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant <i>Aspergillus </i>strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Ecto-Phosphatase Activities on the Cell Surface of the Amastigote Forms of Trypanosoma cruzi

1999 ◽  
Vol 54 (11) ◽  
pp. 977-984 ◽  
Author(s):  
José Roberto Meyer-Fernandes ◽  
Mario Alberto da Silva-Neto ◽  
Mirna dos Santos Soares ◽  
Eloise Fernandes ◽  
Anibal Eugênio Vercesi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Trypanosoma Cruzi ◽  
Phosphatase Activity ◽  
Sodium Fluoride ◽  
Competitive Inhibitor ◽  
Physiological Conditions ◽  
Basal Activity ◽  
Activity Difference ◽  
Pnpp Hydrolysis

Abstract Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phos-pho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hy­drolysed at a rate of 80 nmol ·mg -1 ·h -1 in the presence of 5 mм MgCl2, pH 7.2 at 30 °C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mм MgCl2· Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mм ) . In the absence of Mg2+ (basal activity) the stimulating half concentration (S0. 5) for PNPP was 1.57 mм , while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg2+-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mм . The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phos-phothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-aminoacids and phosphoproteins under physiological conditions.

Interference of ʟ-α-Aminooxy-β-Phenylpropionic Acid with Phenylalanine Metabolism in Buckwheat

1979 ◽  
Vol 34 (12) ◽  
pp. 1162-1173 ◽  
Author(s):  
Heike Holländer ◽  
Hans-Hermann Kiltz ◽  
Nikolaus Amrhein
Keyword(s):  
Amino Acids ◽  
Feedback Control ◽  
Phenylalanine Ammonia Lyase ◽  
Shikimate Pathway ◽  
Fold Increase ◽  
Competitive Inhibitor ◽  
Phenylpropionic Acid ◽  
Enhancing Effect ◽  
Phenylalanine Metabolism ◽  
Effect Of Light

ʟ-α-Aminooxy-β-phenylpropionic acid (AOPP), a potent competitive inhibitor of phenylalanine ammonia-lyase (PAL), blocked light-induced phenylpropanoid synthesis in excised buckwheat hypocotyls and produced an up to 40-fold increase in the endogenous phenylalanine concentra­tion, while the level of all other amino acids was hardly affected. After a 24 h incubation in the light in the presence of 0.3 or 1 mᴍ AOPP phenylalanine alone constituted about 25% of the total soluble amino acids, compared to appr. 1% in the controls. In the presence of AOPP illuminated hypocotyls accumulated nearly 3 times more phenylala­nine than hypocotyls kept in the dark, indicating an enhancing effect of light on the flow of carbon through the shikimate pathway. Exogenously added [14C] phenylalanine was extensively metab­olized by control tissue, but accumulated in AOPP treated tissue. In the presence of AOPP radio­activity from [14C] shikimate accumulated predominantly in phenylalanine, and the flow of shi­kimate into tyrosine and phenylalanine was not affected by the inhibitor. Therefore, under these conditions no feedback control of phenylalanine and tyrosine synthesis from shikimate is apparent in buckwheat hypocotyls.

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