Search results for "enzyme kinetics"

Calcium efflux from human erythrocyte ghosts

1970

The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with(45)Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that(45)Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1MM Ca in the external solution ([Ca(++)] o ), the rate constant of passive Ca efflux (k min(-1), fraction of(45)Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min(-1). There was no further increase at hig…

Epimagnolin A, a tetrahydrofurofuranoid lignan from Magnolia fargesii, reverses ABCB1-mediated drug resistance.

2018

Abstract Background Epimagnolin A is an ingredient of the Chinese crude drug Shin-i, derived from the dried flower buds of Magnolia fargesii and Magnolia flos, which has been traditionally used for the treatment of allergic rhinitis and nasal congestion, empyema, and sinusitis. The pharmacokinetic activity of epimagnolin A remains to be evaluated. Purpose In this study, we examined the possible interactions of epimagnolin A with human ATP-binding cassette (ABC) transporter ABCB1, a membrane protein vital in regulating the pharmacokinetics of drugs and xenobiotics. Study design/methods The interaction of epimagnolin A with ABCB1 was evaluated in calcein, ATPase, and MTT assays by using Flp-I…

Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii

2014

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied t…

The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA

2016

The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ R…

Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13.

2021

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs.To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site.CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The Km-values of the other coumarins varied 0.85-97 µM, Vmax-values of the oxidation reaction varied 0.25-60 min-1, and intrinsic clearance varied 26-…

Differential behaviour of Pseudomonas sp. 42A2 LipC, a lipase showing greater versatility than its counterpart LipA

2009

Abstract Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expr…

Specific, concentration-dependent uptake of vitellin by the oocytes ofNereis virens (Annelida, Polychaeta) in vitro

1991

One purpose of this study was the development of methods for radioactive labeling and handling of the yolk protein of Nereis virens, another, the utilization of this tracer in nereid oocyte cultures to describe the kinetics and specificity of yolk protein uptake. Accumulation of labeled yolk protein by growing oocytes is linear over time, specific for yolk protein, and dependent on tracer concentration in the medium. Incorporation of yolk protein follows saturation kinetics which allows us to reproducibly determine the characteristics of this process. The apparent KM of ∼ 1 × 10−8 M indicates the high affinity of the transport system; the Vmax (160–400 pg protein/day × oocyte) reveals that …

2020

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the SNAr addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly…

Thermal inactivation at high temperatures and regeneration of green asparagus peroxidase

2019

A spectrophotometric method was developed for determining the peroxidase activity of green asparagus in small samples. The optimum conditions for the analysis in the cuvette were 45 mM of H2O2 36 mM of guaiacol, and pH 7. The method can be used to determine enzyme activity at up to two decimal reductions. A study was performed of the regeneration and inactivation kinetics of the enzyme when heated between 90 and 125°C. Regenerated asparagus peroxidase reached its maximum activity after being stored 6 days at 25°C. The regenerated enzyme followed first-order inactivation kinetics, showing an Ea = 13.62 kcal/mol and k100°C = 2.07 min-1.

A hypothetical model of the influence of inorganic phosphate on the kinetics of pyruvate kinase

2000

This paper presents a simple solution to the problem of approximating the calculated curve of reaction progress to the measured curve which is usually disturbed by initial oscillation of auxiliary lactate dehydrogenase (LDH) reaction. The experiments leading to the determination of the apparent Km for phosphoenolpyruvate (PEP) and Vm were performed. For precise estimation of kinetic parameters (Km and Vm) of the M1 isozyme of pyruvate kinase (PK), measured by coupling it to LDH reaction, the sequence of Michaelis‐Menten for pyruvate kinase and second-order kinetics for lactate dehydrogenase reaction as well as a non-zero initial concentration of lactate was assumed. The functions of apparen…