scholarly journals A Simple Protocol for the Determination of Lysostaphin Enzymatic Activity

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 917
Author(s):  
Alexander V. Grishin ◽  
Svetlana V. Konstantinova ◽  
Irina V. Vasina ◽  
Nikita V. Shestak ◽  
Anna S. Karyagina ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Catalytic Efficiency ◽  
Bacterial Cells ◽  
Binding Domains ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
Osmotic Lysis ◽  
Cross Bridges ◽  
Bacterial Peptidoglycan

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

Learning the ABCs one at a time: structure and mechanism of ABC transporters

2019 ◽  
Vol 47 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Robert C. Ford ◽  
Konstantinos Beis
Keyword(s):  
Abc Transporters ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Bacterial Cells ◽  
The Novel ◽  
Mechanism Model ◽  
Binding Domains ◽  
Atp Binding And Hydrolysis ◽  
Alternating Access

Abstract ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.

Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments

2009 ◽  
Vol 131 (42) ◽  
pp. 15251-15256 ◽  
Author(s):  
Jung-uk Shim ◽  
Luis F. Olguin ◽  
Graeme Whyte ◽  
Duncan Scott ◽  
Ann Babtie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Enzymatic Activity ◽  
Simultaneous Determination ◽  
Bacterial Cells

Improved bioluminescent assay of somatic cell counts in raw milk

2008 ◽  
Vol 75 (3) ◽  
pp. 279-283 ◽  
Author(s):  
Valery G Frundzhyan ◽  
Inna M Parkhomenko ◽  
Lubov Y Brovko ◽  
Natalia N Ugarova
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Raw Milk ◽  
Superior Performance ◽  
Bacterial Cells ◽  
Bioluminescent Assay ◽  
Direct Microscopy ◽  
Cell Counts ◽  
Simple Protocol

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1·5% w/w. There was high correlation (R2=0·99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2–3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

A Method for the Colorimetric Determination of β-Glucuronidase in Urine, Serum, Tissue; Assay of Enzymatic Activity in Health and Disease

1959 ◽  
Vol 36 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Julius A. Goldbarg ◽  
Esteban P. Pineda ◽  
Benjamin M. Banks ◽  
Alexander M. Rutenburg
Keyword(s):  
Enzymatic Activity ◽  
Colorimetric Determination ◽  
Health And Disease ◽  
Urine Serum

Bacterial Peptidoglycan Stapling with Functionalized D-Amino Acids

2019 ◽  
Author(s):  
Sylvia L. Rivera ◽  
Akbar Espaillat ◽  
Arjun K. Aditham ◽  
Peyton Shieh ◽  
Chris Muriel-Mundo ◽  
...  
Keyword(s):  
Cell Wall ◽  
Clinical Success ◽  
Bacterial Species ◽  
Enzymatic Reaction ◽  
Amino Acid Derivative ◽  
Wall Structure ◽  
Live Bacteria ◽  
Cross Links ◽  
Osmotic Lysis ◽  
Bacterial Peptidoglycan

Transpeptidation reinforces the structure of cell wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many bacterial species makes it challenging to determine cross-link function precisely. Here we present a technique to covalently link peptide strands by chemical rather than enzymatic reaction. We employ bio-compatible click chemistry to induce triazole formation between azido- and alkynyl-D-alanine residues that are metabolically installed in the cell walls of Gram-positive and Gram-negative bacteria. Synthetic triazole cross-links can be visualized by substituting azido-D-alanine with azidocoumarin-D-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell wall stapling protects the model bacterium Escherichia coli from β-lactam treatment. Chemical control of cell wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.<br>

The Determination of the Noise Pollution in a Production Hall and the Diseases Produced by It

2018 ◽  
Vol 69 (4) ◽  
pp. 875-880
Author(s):  
Oana Roxana Chivu ◽  
Ovidiu Mederle ◽  
Augustin Semenescu ◽  
Ileana Mates ◽  
Claudiu Babis ◽  
...  
Keyword(s):  
Risk Factor ◽  
Human Body ◽  
Noise Level ◽  
Noise Pollution ◽  
Protective Measures ◽  
Work Program ◽  
Specialized Equipment ◽  
Chemical Products ◽  
Main Activity

The paper describes how to determine the noise in a production hall where there are production machines and equipment. The chosen plant is a production unit where filling of the polyurethane foam tubes is performed. The main activity is the production of professional insulators and wholesale of other products from the same range enumerating the following: wholesale chemical products; production of professional insulators; trade in chemicals and chemical industry. The recommended maximum admissible value for a normal 8h work program is 85 dB (A). The objective is to determine the level of noise in the factory and how it acts as a physical professional risk factor and its effects on the human body. The way in which the proposed objective was achieved was to determine the value of the noise level with the specialized equipment. Following the determinations that will be presented in the following chapters, appropriate protective measures have been taken.

Diagnostics of the location of damage to tubing oil well pipes

2019 ◽  
Vol 5 (4) ◽  
pp. 115-128
Author(s):  
Timur A. Ishmuratov ◽  
Rif G. Sultanov ◽  
Milyausha N. Khusnutdinova
Keyword(s):  
Fluid Motion ◽  
Oil Well ◽  
General Belief ◽  
Oil Flow ◽  
Specialized Equipment ◽  
Improving Accuracy ◽  
Analytical Expressions ◽  
Technical Measures ◽  
Quick Determination

The study is devoted to the mathematical description of the process of oil outflow in places of leakage of the tubing string, which allows a computer to locate a leakage in the tubing. The authors propose methodology for identifying defects in the tubing and determining the location of the leak. The uniqueness of this methodology lies in quick determination of the place of leakage without the use of specialists, sophisticated and specialized equipment. Mathematical modeling of oil flow in the tubing requires the apparatus of continuum mechanics. It is a general belief that the movement of oil in the pipe flows at low speeds due to its outflow from the hole. Using the general equations of mass and energy balance, the authors have obtained differential equations of fluid motion in a vertical pipe in the process of its outflow from the tubing and in the process of injection. Analytical expressions are the solution to these equations, as they can help in estimating the degree of damage and its location, as well as the feasibility of its eliminating. The results show that an increase in the leakage and injection times leads to improving accuracy of locating damage. Thus, when conducting various geological and technical measures (GTM) at the well, it is possible to assess the presence of leakage and its intensity when deciding on the repair of tubing.

scholarly journals Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals

2021 ◽  
Vol 11 (2) ◽  
pp. 94
Author(s):  
Masaki Kumondai ◽  
Akio Ito ◽  
Evelyn Marie Gutiérrez Rico ◽  
Eiji Hishinuma ◽  
Akiko Ueda ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Three Dimensional ◽  
Warfarin Dose ◽  
Catalytic Efficiency ◽  
Therapeutic Window ◽  
Wild Type ◽  
Drug Metabolizing Enzyme ◽  
Novel Variants ◽  
Metabolizing Enzyme ◽  
Almost All

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype–phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

Fluorimetric Determination Of Mercury (Ii) Based On The Inhibition Of The Enzymatic Activity Of Urease

1994 ◽  
Vol 27 (5) ◽  
pp. 867-878 ◽  
Author(s):  
D. W. Bryce ◽  
J. M. Fernández-Romero ◽  
M. D. Luque de Castro
Keyword(s):  
Enzymatic Activity ◽  
Fluorimetric Determination

scholarly journals Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Takeo Tomita ◽  
Hajime Matsushita ◽  
Ayako Yoshida ◽  
Saori Kosono ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Glutamate Dehydrogenase ◽  
Ternary Complex ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Regulatory Subunit ◽  
Binary Complex ◽  
Bacterial Cells ◽  
Adenine Phosphoribosyltransferase ◽  
Content Type

ABSTRACT Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

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