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Enzyme kinetics

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https://www.readbyqxmd.com/read/27923176/co-distribution-of-cysteine-cathepsins-and-matrix-metalloproteases-in-human-dentin
#1
Polliana Mendes Candia Scaffa, Lorenzo Breschi, Annalisa Mazzoni, Cristina de Mattos Pimenta Vidal, Rosa Curci, Fabianni Apolonio, Pietro Gobbi, David Pashley, Leo Tjäderhane, Ivarne Luis Dos Santos Tersariol, Fábio Dupart Nascimento, Marcela Rocha Carrilho
It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin...
November 21, 2016: Archives of Oral Biology
https://www.readbyqxmd.com/read/27922051/catalysis-and-structure-of-zebrafish-urate-oxidase-provide-insights-into-the-origin-of-hyperuricemia-in-hominoids
#2
Marialaura Marchetti, Anastasia Liuzzi, Beatrice Fermi, Romina Corsini, Claudia Folli, Valentina Speranzini, Francesco Gandolfi, Stefano Bettati, Luca Ronda, Laura Cendron, Rodolfo Berni, Giuseppe Zanotti, Riccardo Percudani
Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids...
December 6, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27921033/new-tools-to-study-contact-activation
#3
Steffen Rosén
The recent availability of a sensitive chromogenic method approach for determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out. For both enzymes the assay range 1-10 nmol/L provides a resolution of about 0.8 absorbance units with a total assay time of about 20 min. For studies on activation kinetics, subsampling and extensive dilution can be performed in MES-bovine serum albumin (BSA) buffer pH 5.7 for quenching of enzyme activity and with ensuing determination of FXa generation in a chromogenic FXIa method...
2016: Frontiers in Medicine
https://www.readbyqxmd.com/read/27920296/a-sulfur-oxygenase-from-the-haloalkaliphilic-bacterium-thioalkalivibrio-paradoxus-with-atypically-low-reductase-activity
#4
Patrick Rühl, Uwe Pöll, Johannes Braun, Andreas Klingl, Arnulf Kletzin
: Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 (TpSOR) is branching deeply within dendrograms of these proteins (29-34% identity). A synthetic gene encoding the TpSOR expressed in E. coli resulted in a protein of 14.7±0.9 nm in diameter and an apparent molecular mass of 556 kDa. Sulfite and thiosulfate were formed from elemental sulfur in a temperature range of 10-98°C (Topt≈80°C) and a pH range of 6-11...
December 5, 2016: Journal of Bacteriology
https://www.readbyqxmd.com/read/27919007/challenges-in-assignment-of-allosteric-effects-in-cytochrome-p450-catalyzed-substrate-oxidations-to-structural-dynamics-in-the-hemoprotein-architecture
#5
Peter Hlavica
Cytochrome P450s (CYP) represent a superfamily of b-type hemoproteins catalyzing NAD(P)H-dependent oxidative biotransformation of a vast array of natural and xenobiotic compounds. Many eu- and prokaryotic members of this class of monooxygenases display complex non-Michaelis-Menten saturation kinetics, suggestive of homo-/heterotropic cooperativity arising from substrate-/effector-induced allosteric interactions. Here, the paradigm of multiple-ligand occupancy of the catalytic pocket in combination with enzyme oligomerization provides the most favored explanations for the atypical kinetic patterns...
November 25, 2016: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/27918060/effect-of-small-molecules-on-cell-reprogramming
#6
REVIEW
M Baranek, A Belter, M Z Naskręt-Barciszewska, M Stobiecki, W T Markiewicz, J Barciszewski
The essential idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are able to indefinitely self-renew and differentiate into all cell types of the body which makes them a potent substantial player in regenerative medicine. The easily accessible source of induced pluripotent stem cells may allow obtaining and cultivating tissues in vitro. Reprogramming refers to regression of mature cells to its initial pluripotent state. One of the approaches affecting pluripotency is the usage of low molecular mass compounds that can modulate enzymes and receptors leading to the formation of pluripotent stem cells (iPSCs)...
December 5, 2016: Molecular BioSystems
https://www.readbyqxmd.com/read/27917251/droplet-millifluidics-for-kinetic-study-of-transketolase
#7
A Pinsolle, F Charmantray, L Hecquet, F Sarrazin
We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme...
November 2016: Biomicrofluidics
https://www.readbyqxmd.com/read/27916677/differential-copper-binding-to-alpha-synuclein-and-its-disease-associated-mutants-affect-the-aggregation-and-amyloid-formation
#8
Priyatosh Ranjan, Dhiman Ghosh, Deepthi S Yarramala, Subhadeep Das, Samir K Maji, Ashutosh Kumar
BACKGROUND: Copper is an essential trace element required for the proper functioning of various enzymes present in central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurogenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu(2+) to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. METHODS: We investigated the residue specific mapping of Cu(2+) binding sites and binding strength using solution-state NMR and ITC respectively...
December 1, 2016: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/27912158/direct-detection-of-phenylephrine-3-o-sulfate-in-ls180-human-intestinal-cells-using-a-novel-hydrophilic-interaction-liquid-chromatography-hilic-assay
#9
Heta N Shah, Matthew T Halquist, Phillip M Gerk
The efficacy of phenylephrine (PE) is controversial due to its extensive pre-systemic metabolism through sulfation to form phenylephrine-3-O-sulfate (PES). Hence quantitation of PES is important in order to study the metabolism of PE. There are no published methods available for direction detection of PES. We have developed and validated a hydrophilic interaction liquid chromatography (HILIC) method for the direct detection of PES and simultaneous detection of PE to study the enzyme kinetics and metabolism of PE to enable approaches to reduce the presystemic metabolism of PE...
November 13, 2016: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/27911826/structural-insights-into-the-lcib-protein-family-reveals-a-new-group-of-%C3%AE-carbonic-anhydrases
#10
Shengyang Jin, Jian Sun, Tobias Wunder, Desong Tang, Asaph B Cousins, Siu Kwan Sze, Oliver Mueller-Cajar, Yong-Gui Gao
Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-inducible B protein (LCIB) is known to be essential for CCM function in Chlamydomonas reinhardtii To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs...
December 1, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27910729/identification-of-cytochrome-p450s-involved-in-the-metabolism-of-6-benzyl-1-benzyloxymethyl-5-iodouracil-w-1-using-human-recombinant-enzymes-and-rat-liver-microsomes-in-vitro
#11
Ying-Yuan Lu, Hai-Xu Cheng, Xin Wang, Xiao-Wei Wang, Jun-Yi Liu, Pu Li, Ya-Qing Lou, Jun Li, Chuang Lu, Guo-Liang Zhang
1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTIs) in rat and human in vitro. 2. The parent drug of W-1 was incubated with RLMs or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5, respectively) in the presence or absence of NADPH regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS)...
December 2, 2016: Xenobiotica; the Fate of Foreign Compounds in Biological Systems
https://www.readbyqxmd.com/read/27909846/benzimidazole-inhibitors-of-protein-kinase-ck2-potently-inhibit-the-activity-of-atypical-protein-kinase-rio1
#12
Konrad Kubiński, Maciej Masłyk, Andrzej Orzeszko
Benzimidazole derivatives of 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) comprise the important class of protein kinase CK2 inhibitors. Depending on the structure, benzimidazoles inhibit CK2 with different selectivity and potency. Besides CK2, the compounds can inhibit, with similar activity, other classical eukaryotic protein kinases (e.g. PIM, DYRK, and PKD). The present results show that a majority of the most common CK2 inhibitors can affect the atypical kinase Rio1 in a nanomolar range. Kinetic data confirmed the mode of action of benzimidazoles as typical ATP-competitive inhibitors...
December 1, 2016: Molecular and Cellular Biochemistry
https://www.readbyqxmd.com/read/27908259/a-novel-liquid-chromatography-tandem-mass-spectrometry-method-for-the-estimation-of-bilirubin-glucuronides-and-its-application-to-in-vitro-enzyme-assays
#13
Siva Prasad Putluru, Murali Krishna Matta, Deepak Ahire, Murali Subramanian, Michael Sinz, Sandhya Mandlekar
Bilirubin is a toxic waste product of metabolism, eliminated mainly through UGT1A1 mediated conjugation to mono- and di-glucuronides. Due to the potentially low Km value of bilirubin glucuronidation, the quantitative sensitivity obtained with most UV/visible light detection methods are not sufficient to accurately calculate UGT1A1 enzyme kinetics and inhibition properties of new chemical entities at low bilirubin concentrations. In addition, bilirubin, as well as its metabolites, are unstable during sample preparation and bioanalysis...
November 24, 2016: Drug Metabolism Letters
https://www.readbyqxmd.com/read/27908241/molecular-cloning-biochemical-characterization-and-antitumor-properties-of-a-novel-l-asparaginase-from-synechococcus-elongatus-pcc6803
#14
R Kebeish, A El-Sayed, H Fahmy, A Abdel-Ghany
L-asparaginase (EC 3.5.1.1), which catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia, has been widely used as a key therapeutic tool in the treatment of tumors. The current commercially available L-asparaginases, produced from bacteria, have signs of toxicity and hypersensitivity reactions during the course of tumor therapy. Therefore, searching for L-asparaginases with unique biochemical properties and fewer adverse effects was the objective of this work. In this study, cyanobacterial strain Synechococcus elongatus PCC6803 was found as a novel source of L-asparaginase...
October 2016: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/27908238/thermodynamic-analysis-of-fast-stages-of-specific-lesion-recognition-by-dna-repair-enzymes
#15
REVIEW
N A Kuznetsov, O S Fedorova
The methodology of determination of the thermodynamic parameters of fast stages of recognition and cleavage of DNA substrates is described for the enzymatic processes catalyzed by DNA glycosylases Fpg and hOGG1 and AP endonuclease APE1 during base excision repair (BER) pathway. For this purpose, stopped-flow pre-steady-state kinetic analysis of tryptophan fluorescence intensity changes in proteins and fluorophores in DNA substrates was performed at various temperatures. This approach made it possible to determine the changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA-substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage...
October 2016: Biochemistry. Biokhimii︠a︡
https://www.readbyqxmd.com/read/27908109/perturbation-of-hydration-layer-in-solvated-proteins-by-external-electric-and-electromagnetic-fields-insights-from-non-equilibrium-molecular-dynamics
#16
Prithwish K Nandi, Zdenek Futera, Niall J English
Given the fundamental role of water in governing the biochemistry of enzymes, and in regulating their wider biological activity (e.g., by local water concentration surrounding biomolecules), the influence of extraneous electric and electromagnetic (e/m) fields thereon is of central relevance to biophysics and, more widely, biology. With the increase in levels of local and atmospheric microwave-frequency radiation present in modern life, as well as other electric-field exposure, the impact upon hydration-water layers surrounding proteins, and biomolecules generally, becomes a particularly pertinent issue...
November 28, 2016: Journal of Chemical Physics
https://www.readbyqxmd.com/read/27907199/identification-heterologous-expression-and-functional-characterization-of-bacillus-subtilis-yutf-a-had-superfamily-5-nucleotidase-with-broad-substrate-specificity
#17
Natalia P Zakataeva, Dmitriy V Romanenkov, Yuliya R Yusupova, Victoria S Skripnikova, Takayuki Asahara, Sergey V Gronskiy
5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools...
2016: PloS One
https://www.readbyqxmd.com/read/27906638/gene-duplications-and-losses-among-vertebrate-deoxyribonucleoside-kinases-of-the-non-tk1-family
#18
Zeeshan Mutahir, Louise Slot Christiansen, Anders R Clausen, Martin W Berchtold, Zoran Gojkovic, Birgitte Munch-Petersen, Wolfgang Knecht, Jure Piškur
Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK)...
December 2016: Nucleosides, Nucleotides & Nucleic Acids
https://www.readbyqxmd.com/read/27906629/purification-activity-and-expression-levels-of-two-uridine-cytidine-kinase-isoforms-in-neuroblastoma-cell-lines
#19
R Meinsma, A B P van Kuilenburg
Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme...
December 2016: Nucleosides, Nucleotides & Nucleic Acids
https://www.readbyqxmd.com/read/27906051/the-lysine-methyltransferase-ehmt2-g9a-is-dispensable-for-skeletal-muscle-development-and-regeneration
#20
Regan-Heng Zhang, Robert N Judson, David Y Liu, Jürgen Kast, Fabio M V Rossi
BACKGROUND: Euchromatic histone-lysine N-methyltransferase 2 (G9a/Ehmt2) is the main enzyme responsible for the apposition of H3K9 di-methylation on histones. Due to its dual role as an epigenetic regulator and in the regulation of non-histone proteins through direct methylation, G9a has been implicated in a number of biological processes relevant to cell fate control. Recent reports employing in vitro cell lines indicate that Ehmt2 methylates MyoD to repress its transcriptional activity and therefore its ability to induce differentiation of activated myogenic cells...
May 27, 2016: Skeletal Muscle
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