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The Renin-Angiotensin System
Published in Austin E. Doyle, Frederick A. O. Mendelsohn, Trefor O. Morgan, Pharmacological and Therapeutic Aspects of Hypertension, 2020
There is some controversy as to the effect of circulating levels of plasma-renin substrate on its reaction rate with renin. A direct way of studying this problem is to measure both the kinetics of the reaction and circulating levels of substrate. This allows calculation of the effect of substrate concentration on reaction velocity using the Michaelis-Menten equation. However, the results reported using this approach in man have been very variable because of the wide range of values obtained for both circulating substrate and the marked discrepancies in the published values of the Km of the reaction, which vary over a 20- to 50-fold range.140,148,149,170,187,198,199 However, the results obtained for Km using purified substrate suggests that plasma-substrate levels are important determinants of reaction rates. This conclusion is supported by the fact that many groups have observed that the rate of formation of angiotensin I during in vitro incubation of human plasma (“PRA”) is increased in samples with elevated substrate concentrations.171,187,191,194,200
Methods of Protein Iodination
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
The reagent, when incubated with a protein, forms amidines with protein amines the way illustrated for a lysyl ϵ-amino group in Figure 15 (see Structure 6). Wood and associates140 studied the reactivity of albumins (bovine, canine) with the compound. On the whole, a much slower reactivity of the imidoester as compared to the carboxylic acid ester used by Bolton and Hunter135 was prominent. The reaction time was measured in hours (24 hr), instead of minutes. At a molar ester-to-albumin ratio of 14 and an albumin concentration of 20 mg/m𝓁, the maximal rate of incorporation of radioactivity was 1 to 2% per hour, with a maximal incorporation of 30% of the 125I present. In the pH range of 6.5 to 9.5, the reaction velocity doubled when the pH was increased by 1.4 units. Temperature affected the reaction so that a 10°C-increase between 0 and 40°C tripled the reaction rate. Furthermore, the reaction velocity increased linearly as a function of the concentrations of both the imidoester and the albumin.
Physiology, Biochemistry, and Pathology of Neuromuscular Transmission
Published in Marc H. De Baets, Hans J.G.H. Oosterhuis, Myasthenia Gravis, 2019
Figure 10 shows the reaction scheme of the hydrolysis of substrates by the catalytic center of the AChE. There are vast differences in reaction velocity between substrates, such as ACh itself which is hydrolyzed in much less than a millisecond, and “poisoning” substrates, such as pyridostigmine that is processed by the enzyme in about 5,000 seconds.
Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Krzysztof M. Zak, Mark J. Bostock, Irena Waligorska, Ida B. Thøgersen, Jan J. Enghild, Grzegorz M. Popowicz, Przemyslaw Grudnik, Jan Potempa, Miroslaw Ksiazek
Mirolysin (6 nM) was incubated at 37 °C in 0.1 M Tris, 0.15 M NaCl, 5 mM CaCl2, 0.05% Pluronic-F127, 0.02% NaN3, pH 7.5 in the presence of increasing concentrations of cpd 9 (0–7.2 µM) and cpd 10 (0–38.4 µM) in microtiter plate wells (total volume, 100 µl). After 15 min of incubation at 37 °C, 100 µl fluorogenic protein substrate FTC-casein was added at different concentrations (5–120 µg/ml), and the residual proteolytic activity was recorded. The mode of inhibition of target proteases by the compounds was determined graphically using the Lineweaver–Burk plot using Equation (2): V is the reaction velocity, Vmax is the maximum V, Km is the Michaelis–Menten constant, and [S] is the substrate concentration. The Ki was determined using a macro for competitive inhibition from GraphPad Prism software (La Jolla, CA, USA) and Equation (3): I] is the inhibitor concentration.
Refining the structure−activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Joana Magalhães, Nina Franko, Giannamaria Annunziato, Marco Pieroni, Roberto Benoni, Anna Nikitjuka, Andrea Mozzarelli, Stefano Bettati, Anna Karawajczyk, Aigars Jirgensons, Barbara Campanini, Gabriele Costantino
Enzyme activity under steady-state conditions was measured by a discontinuous method as described previously23. Briefly, the reaction was carried out in 100 mM HEPES pH 7.0 in the presence of 6 nM OASS, 60 nM BSA to prevent enzyme adhesion to tube wall, 1 mM OAS (equal to the Km value of this subsrate), 0.6 mM Na2S (a saturating concentration), 1% DMSO and variable concentrations of inhibitor. Reaction was stopped at time intervals by the addition of acetic acid and concentration of cysteine was determined by a modification of the ninhydrin assay25. The fractional velocity as a function of inhibitor concentration was determined and IC50 was calculated by the following equation: v0 is the reaction velocity in the absence of inhibitor and vi is reaction velocity in the presence of inhibitor at concentration [I].
Predominant contributions of carboxylesterase 1 and 2 in hydrolysis of anordrin in humans
Published in Xenobiotica, 2018
Jinfang Jiang, Xiaoyan Chen, Dafang Zhong
The parent depletion method was used to determine kinetics of anordrin hydrolysis in different enzyme sources. In preliminary study, we confirmed linear depletion rate of anordrin with respect to protein concentrations (<1.5 mg/mL HLM and HIM, <1.0 mg/mL CES1 and CES2) and incubation time (<60 min in HLM and HIM, <2 h in CES1 and CES2). For kinetic analysis, selected final conditions were as follows: HLM: 0.1 mg/mL, 10 min; HIM: 0.2 mg/mL, 40 min; CES1 and CES2: 0.2 mg/mL, 30 min. Anordrin concentration ranged from 0.1 to 20 μM. Incubation procedure was similar to method described for anordrin hydrolysis activity. Control samples were incubated with thermally inactivated enzymes. Data are presented as averages of triplicate experiments. Kinetic constants were obtained by fitting Michaelis–Menten equation shown in Equation 1 to experimental data by using nonlinear regression (Prism 5.0; GraphPad Software Inc., La Jolla, CA): v is reaction velocity, Vmax is maximum velocity, Km is Michaelis constant (substrate concentration at 0.5Vmax) and S is substrate concentration. In vitro CLint was calculated as Vmax/Km.