enzyme assay
Recently Published Documents


TOTAL DOCUMENTS

724
(FIVE YEARS 97)

H-INDEX

47
(FIVE YEARS 3)

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuexiang Lin ◽  
Xiao-Yu Liu ◽  
Bo Zhang ◽  
Ai-Qing Qin ◽  
Kwok-Min Hui ◽  
...  

AbstractCurrent methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66–85.84%) and 95.39% (95% confidence interval: 94.32–96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.


Author(s):  
Mamidala Shyam Prasad ◽  
Muralidhar P. Ande ◽  
Karthireddy Syamala ◽  
Narinder Kumar Chadha ◽  
Paramita Banerjee Sawant ◽  
...  

Background: Stunting is a process of suppressing growth from unfavourable conditions. The protein supplementation during stunting gives scope to maintain the nutrient reserves of fish and its quality. Methods: A feeding trial was conducted for eight months to study the effect of three hetero-nitrogenous diets with 25% (control), 30% (T1) and 35% (T2) crude protein (CP) levels on growth and physio-metabolic responses of Chanos chanos fingerlings during stunting. Milk fish fingerlings with a mean body weight of 11.71±0.18 g were stocked in earthen ponds @ 20 no/m2 in each replicate (n=3) was fed @ 2% biomass throughout the experiment. Result: Fish fed with T1 diet showed better specific growth rate (0.64±0.01% d-1), weight gain percentage (362.56±14.95) and protease activity (7.53±0.25 U/mg protein). Whereas, lower activity was observed for the enzyme assay, namely superoxide dismutase (45.41±2.50 U/min/mg protein), aspartate aminotransferase (34.01±1.88 U/min/mg protein) and alanine aminotransferase (39.64±0.64 U/min/mg protein). Hence, it may be concluded that the dietary protein inclusion level of 30% CP showed better growth performance and lower physio-metabolic response in milkfish fingerlings during the stunting.


Author(s):  
Romică CREȚU

In the last decade studies show that water pollution is a very serious problem, especially since the degree of water pollution plays a key role in the growth and fish multiplication. As a biomarker of environmental pollution, antioxidant enzymes such as catalase (CAT; EC 1.11.1.6) play an essential role in preventing the harmful effects of heavy metals in the tissues of fish. These researches were carried out to investigate the effect of various environment conditions and some pollutant agents on oxidative stress in some aquatic organisms. The enzymatic assay of CAT (in fish organs, e.g., liver, kidney, gill, intestine and brain, as well as in mixture of these organs) was carried out according to standard enzyme assay protocol. The results showed decrease of CAT activity: the enzymatic activity was 35.89 ± 1.02 µmol H2O2/min/mg protein to pH 7 and 6.59 ± 0.47 µmol H2O2/min/mg protein to pH 12. More, the enzymatic activity was 38.1 ± 0.3 µmol H2O2/min/mg protein to pH 3. Also, the catalase activity increased with 23 enzymatic units to a less dissolved oxygen concentration (6.2877 mg/L). Furthermore, this study indicated that heavy metals, such as Cr, Cu and Zn can inhibit biochemical reactions in various organs of fish. During exposure duration of the fish to a mixture of metal ions the catalase activity decreases from 35.89 ± 1.02 µmol H2O2/min/mg protein to 23.51 ± 2.85 µmol H2O2/min/mg protein. On the other hand, as a result of the response of the enzyme system, the catalase activity increases to 36.25 ± 3.22 µmol H2O2/min/mg protein.


2021 ◽  
Author(s):  
◽  
Nisha Das ◽  

Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to modify and inactivate amSPCs has been examined by two unique biochemical assays, including an agar-based enzyme assay. Binding of APH (9)-Ia and ANT (3")(9) to spectinomycin and amSPCs has been studied using Thermal Denaturation assay and MicroScale Thermophoresis (MST). The microbiological role of these enzymes has been examined by minimum inhibitory concentration (MIC) shifts using an arabinose inducible expression of APH (9)-Ia and ANT (3")(9) in E.coli K12 and JW ΔtolC strains. Our agar-based enzyme assay shows the inactivation of spectinomycin by APH(9)-Ia. Phosphorylated spectinomycin and adenylated spectinomycin products upon incubation with APH(9)-Ia and ANT(3",9), respectively, have been identified using MALDI-MS. APH(9)-Ia induction studies in E. coli tolC knock-out strains reveal a MIC increase against spectinomycin in the presence of 2% arabinose compared to no shift with amSPCs. ANT (3")(9) showed an increase in MIC against spectinomycin as well as amSPCs. In conclusion, amSPCs are not inactivated by APH (9)-Ia in vivo but are inactivated by ANT (3")(9). Most Gram-negative bacteria isolated in clinics possess one or more AMEs. By overcoming modification by AMEs, amSPCs can be a valuable tool in overcoming resistance in Gram-negative bacterial infections. We also conducted a high throughput screen of a polar small molecule library against two multi-drug resistant clinical isolates of Escherichia coli that encode aminoglycoside modifying enzyme for small molecule potentiators of amSPCs to yield 12 possible potentiating molecules that have been confirmed by dose-response analysis. Future work as a continuation of this project will involve further analysis of any existing synergy between the potentiating molecules and amSPCs and target validation of these potentiators.


2021 ◽  
Vol 14 ◽  
Author(s):  
Shannon Robin ◽  
Khalil Ben-Hassine ◽  
Simona Jurkovic Mlakar ◽  
Vid Mlakar ◽  
Marc Ansari ◽  
...  

Background: Glutathione S-transferases (GSTs) are phase II metabolic enzymes crucial for the metabolism of electrophilic drugs. Additionally, several GST isoforms are involved in protein-protein interaction with mitogen-activated protein kinases (MAPKs), modulating apoptosis pathways. Methods: To assess the potential change of enzymatic activity, we performed a GST enzyme assay with human recombinant GSTM1 in the presence and absence of MAPK8. Recently, GSTM1 has been demonstrated to interact with MAPK8 both in silico and in vitro. The binding interface predicted in silico comprised amino acid residues present on the surface of the protein and a few were deep in the active site of the protein. Results: The experiment demonstrated that the GSTM1 activity was conserved even in the presence of MAPK8 in the assay. Conclusion: Thus, this interaction with MAPK8 may potentially cause an alteration of the catalytic activity of GSTM1.


Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1732
Author(s):  
Fiona F. Hager-Mair ◽  
Cordula Stefanović ◽  
Charlie Lim ◽  
Katharina Webhofer ◽  
Simon Krauter ◽  
...  

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sachio Shibata ◽  
Satoshi Sogabe ◽  
Masanori Miwa ◽  
Takuya Fujimoto ◽  
Nobuyuki Takakura ◽  
...  

AbstractLactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.


2021 ◽  
pp. 100141
Author(s):  
Keisuke Kitakaze ◽  
Kazuhito Tsuboi ◽  
Maho Tsuda ◽  
Yasuhiro Takenouchi ◽  
Hironobu Ishimaru ◽  
...  
Keyword(s):  

2021 ◽  
Vol 17 ◽  
Author(s):  
Nafiseh Karimi ◽  
Rouhollah Vahabpour Roudsari ◽  
Zahra Hajimahdi ◽  
Afshin Zarghi

Background: Integrase enzyme is a validated drug target to discover novel structures as anti-HIV-1 agents. Objective: Novel series of thioimidazolyl diketo acid derivatives characterizing various substituents at N-1 and 2-thio positions of central ring were developed as HIV-1 integrase inhibitors. Results: The obtained molecules were evaluated in the enzyme assay, displaying promising integrase inhibitory activity with IC50 values ranging from 0.9 to 7.7 M. The synthesized compounds were also tested for antiviral activity and cytotoxicity using HeLa cells infected by the single-cycle replicable HIV-1 NL4-3. Conclusion: The most potent compound was 18i with EC50=19 µM, IC50 0.9 µM and SI= 10.5. Docking studies indicated that the binding mode of the active molecule is well aligned with the known HIV-1 integrase inhibitors.


Sign in / Sign up

Export Citation Format

Share Document