Studies on the kinetics and regulation of glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 439-450 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Forward Direction ◽  
Product Inhibition ◽  
Reverse Direction ◽  
High Concentrations ◽  
Glutamate Synthesis ◽  
Inhibition Studies

A 190-fold purified preparation of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium was used for the determination of kinetic parameters of the substrates, NADPH, NH4+, and α-ketoglutarate, in the direction of glutamate synthesis, and NADP+ and glutamate in the reverse direction. The kinetic constants determined from this study suggest a biosynthetic role for the enzyme. Based on an analysis of the data derived from initial velocity and product inhibition studies, the reaction mechanism was postulated to be ordered Ter Bi with NADPH as the first substrate to bind in the forward direction, and NADP+ binding first in the reverse direction.Of the several metabolites tested for a possible function in the regulation of GDH activity, only L-malate and L-glutamine appeared to exert an appreciable influence on the enzyme. ATP and AMP at a concentration of 0.8 mM were found to enhance GDH activity by 68% and 6%, respectively, but at high concentrations, both the adenine nucleotides proved to be inhibitory.

Glutamate Dehydrogenase from Medicago sativa L., Purification and Comparative Kinetic Studies of the Organ-Specific Multiple Forms

1980 ◽  
Vol 35 (5-6) ◽  
pp. 406-415 ◽  
Author(s):  
Marianne Nagel ◽  
Hartmann
Keyword(s):  
Medicago Sativa ◽  
Glutamate Dehydrogenase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Enzyme Preparations ◽  
Organ Specific ◽  
Nad Oxidoreductase ◽  
Medicago Sativa L ◽  
Inhibition Studies

Abstract NAD-specific glutamate dehydrogenase [L-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-pattems of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measure­ ments and product inhibition studies are consistent with an ordered ternary-binary kinetic mecha­ nism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.

scholarly journals Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase

1987 ◽  
Vol 242 (1) ◽  
pp. 143-150 ◽  
Author(s):  
K S De Jongh ◽  
P J Schofield ◽  
M R Edwards
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Free Enzyme ◽  
Aldehyde Reductase ◽  
Binding Studies ◽  
Sheep Liver ◽  
Low Concentrations ◽  
High Concentrations ◽  
Inhibition Studies

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.

scholarly journals Kinetic studies on the two common inherited forms of human erythrocyte adenylate kinase

1972 ◽  
Vol 130 (3) ◽  
pp. 805-811 ◽  
Author(s):  
C. Brownson ◽  
N. Spencer
Keyword(s):  
Human Erythrocyte ◽  
Adenylate Kinase ◽  
Competitive Inhibition ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Product Inhibition ◽  
Reverse Direction ◽  
Kinetic Properties ◽  
Ping Pong ◽  
Variable Substrate

1. The kinetic properties of two genetic variants of human erythrocyte adenylate kinase were studied at limiting concentrations of both ADP and MgADP- in the forward direction and at limiting concentrations of both AMP and MgATP2- in the reverse direction. 2. Primary reciprocal plots rule out the possibility of a Ping Pong mechanism for both forms of the enzyme. 3. Analysis of the kinetic data by an appropriate computer program gave the following Km values for the type 1 enzyme: AMP, 0.33mm±0.1; MgATP2-, 0.95mm±0.13; ADP, 0.12mm±0.03; MgADP-, 0.22mm±0.04. Values for the type 2 enzyme were: AMP, 0.27mm±0.03; MgATP2-, 0.40mm±0.05; ADP, 0.08mm±0.07; MgADP-, 0.20mm±0.04. 4. Product inhibition studies were done by studying the reverse reaction. With ADP as product inhibitor competitive inhibition patterns were obtained with AMP and/or MgATP2- as variable substrate. Similar results were obtained for product inhibition by MgADP- with AMP as variable substrate. The results are consistent with a Rapid Equilibrium Random mechanism. 5. Secondary plots of slope versus product concentration were linear. The data were fitted to the appropriate equation and analysed by computer to give values for the product inhibition constants. 6. Differences between the values of certain kinetic constants for the two forms of the enzyme were observed.

Studies on isocitrate lyase from the facultative autotroph Thiobacillus novellus

1973 ◽  
Vol 19 (4) ◽  
pp. 513-519 ◽  
Author(s):  
J. T. McCarthy ◽  
A. Michael Charles
Keyword(s):  
Isocitrate Lyase ◽  
Adenine Nucleotides ◽  
Potassium Phosphate ◽  
Forward Direction ◽  
Disc Electrophoresis ◽  
Reverse Direction ◽  
K Value ◽  
Absolute Requirement ◽  
Sigmoidal Kinetics ◽  
Hill Plots

Isocitrate lyase from Thiobacillus novellus was purified about eightfold from crude extracts of acetate-grown cells. Polyacrylamide gel "disc" electrophoresis revealed that the partly purified material contained one major and two minor peaks. The optimum pH for activity in the forward direction with glycylglycine buffer and the reverse direction with either glycylglycine or potassium phosphate was around pH 8.0. Plots of v vs. s gave Michaelis–Menten kinetics with an apparent Km of 4.3 × 10−5 M for isocitrate and 2.3 × 10−4 M for succinate. However, for glyoxylate, v vs. s plots were sigmoidal. Hill plots gave an n value of 2.6 and a k value of about 7.76 × 10−5 M. Since biosynthetic enzymes usually have sigmoidal kinetics, it follows that isocitrate lyase may be important in the synthesis of isocitrate. Also of interest was the finding that the enzyme did not show an absolute requirement for a divalent cation, but in the presence of 0.3 mM Mg2+ activity was stimulated about threefold over that observed in the absence of metal. Km for Mg2+ was 7.0 × 10−4 M. Complete loss of activity found on addition of 0.03 mM HgCl2 or CuSO4 to the enzyme in the absence of Mg2+, as well as the restoration of activity lost during storage by the addition of cysteine or glutathione supports the idea that sulfhydryl groups are required for activity. The effect of adenine nucleotides on activity revealed in decreasing order of inhibition that ATP > ADP > AMP. Adenosine or pyrophosphate were not inhibitory at any concentration.

Determination of antibacterial propertiesof triazines in regards to Salmonella typhimurium

2017 ◽  
Vol 19 (1) ◽  
pp. 16-23
Author(s):  
M. M. Babkina ◽  
◽  
O. V. Vasylchenko ◽  
O. M. Deriabin ◽  
A. A. Tarasov ◽  
...  
Keyword(s):  
Salmonella Typhimurium

Methods for the Determination of the Purity of Exosomes

2020 ◽  
Vol 25 (42) ◽  
pp. 4464-4485 ◽  
Author(s):  
Katarzyna Kluszczyńska ◽  
Liliana Czernek ◽  
Wojciech Cypryk ◽  
Łukasz Pęczek ◽  
Markus Düchler
Keyword(s):  
Drug Transport ◽  
Biological Fluids ◽  
Drug Carriers ◽  
Biological Functions ◽  
Isolation And Purification ◽  
Pubmed Database ◽  
High Concentrations ◽  
Targeted Release

Background: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance. Methods: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database. Results: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols. Conclusion: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

scholarly journals Electroanalytical Techniques for the Detection of Selenium as a Biologically and Environmentally Significant Analyte—A Short Review

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1768
Author(s):  
Miroslav Rievaj ◽  
Eva Culková ◽  
Damiána Šandorová ◽  
Zuzana Lukáčová-Chomisteková ◽  
Renata Bellová ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Essential Element ◽  
Analytical Techniques ◽  
High Sensitivity ◽  
Stripping Voltammetry ◽  
Short Review ◽  
Electroanalytical Methods ◽  
High Concentrations ◽  
Speed Of Analysis

This short review deals with the properties and significance of the determination of selenium, which is in trace amounts an essential element for animals and humans, but toxic at high concentrations. It may cause oxidative stress in cells, which leads to the chronic disease called selenosis. Several analytical techniques have been developed for its detection, but electroanalytical methods are advantageous due to simple sample preparation, speed of analysis and high sensitivity of measurements, especially in the case of stripping voltammetry very low detection limits even in picomoles per liter can be reached. A variety of working electrodes based on mercury, carbon, silver, platinum and gold materials were applied to the analysis of selenium in various samples. Only selenium in oxidation state + IV is electroactive therefore the most of voltammetric determinations are devoted to it. However, it is possible to detect also other forms of selenium by indirect electrochemistry approach.

scholarly journals The Double Face of Ketamine—The Possibility of Its Identification in Blood and Beverages

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 813
Author(s):  
Magdalena Świądro ◽  
Paweł Stelmaszczyk ◽  
Irena Lenart ◽  
Renata Wietecha-Posłuszny
Keyword(s):  
Date Rape ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Heroin Addiction ◽  
Assisted Extraction ◽  
Low Concentrations ◽  
High Concentrations ◽  
Rosé Wine ◽  
Tof Ms

The purpose of this study was to develop and validate a high-sensitivity methodology for identifying one of the most used drugs—ketamine. Ketamine is used medicinally to treat depression, alcoholism, and heroin addiction. Moreover, ketamine is the main ingredient used in so-called “date-rape” pills (DRP). This study presents a novel methodology for the simultaneous determination of ketamine based on the Dried Blood Spot (DBS) method, in combination with capillary electrophoresis coupled with a mass spectrometer (CE-TOF-MS). Then, 6-mm circles were punched out from DBS collected on Whatman DMPK-C paper and extracted using microwave-assisted extraction (MAE). The assay was linear in the range of 25–300 ng/mL. Values of limits of detection (LOD = 6.0 ng/mL) and quantification (LOQ = 19.8 ng/mL) were determined based on the signal to noise ratio. Intra-day precision at each determined concentration level was in the range of 6.1–11.1%, and inter-day between 7.9–13.1%. The obtained precision was under 15.0% (for medium and high concentrations) and lower than 20.0% (for low concentrations), which are in accordance with acceptance criteria. Therefore, the DBS/MAE/CE-TOF-MS method was successfully checked for analysis of ketamine in matrices other than blood, i.e., rose wine and orange juice. Moreover, it is possible to identify ketamine in the presence of flunitrazepam, which is the other most popular ingredient used in DRP. Based on this information, the selectivity of the proposed methodology for identifying ketamine in the presence of other components of rape pills was checked.

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