Utilization of Automation for Studies of Enzyme Kinetics

Schwartz, Morton K.; Bodansky, Oscar

doi:10.1002/9780470110348.ch3

Cited by 16 publications

(2 citation statements)

References 34 publications

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“…LDH activity was determined spectrophotometrically by monitoring the conversion of NAD + to NADH in a lactate-utilizing reaction [47]. Cellular ATP was assessed using a system based on firefly (Photinus pyralis) luciferase (PerkinElmer ATPLite) using a VICTOR3 multilabel reader (PerkinElmer).…”

Section: Methodsmentioning

confidence: 99%

Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis

et al. 2012

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BackgroundClotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles.Methodology/Principal FindingsThree cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with Ki values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231.Conclusions/SignificanceClotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.

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Section: Methodsmentioning

confidence: 99%

Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis

et al. 2012

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“…Testing the effectiveness and non‐perturbing nature of the quenching agent is an important step to performing quenched assays. Borate buffer and sodium carbonate are usually included in assays to stop the enzymatic reaction by means of a rapid pH change 11 . The study of the effect of both stopping agents in the assay is required to avoid residual activity and to increase experimental accuracy.…”

Section: Introductionmentioning

confidence: 99%

Sodium carbonate versus borate buffer for lactase quenching, laboratory work

Ramos‐de‐la‐Peña

Valenzo

Clorio-Carrillo

et al. 2021

Biochem Molecular Bio Educ

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A laboratory exercise for undergraduate advanced students of enzymology and biocatalysis is presented. Since enzyme assays can be quenched or continuous, this experiment compares the performance of two quenching agents for lactase, in a continuous setup. Enzymatic activity of β‐galactosidase (Aspergillus oryzae) was determined based on the release of 4‐nitrophenol from 4‐nitrophenyl β‐D‐galactopyranoside using a microplate reader. Sodium carbonate and borate buffer were tested as quenching agents, and experimental control was the unstopped assay. Based on released 4‐nitrophenol, enzyme activity, and rate constant k, the students could assess the performance of each termination agent. The experiment promotes disciplinary and transversal competencies, including research‐based learning, critical thinking, and introduce the students to high‐throughput techniques that are common in the research and development environment.

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Generation of continuous ligand–macromolecule binding isotherms. Use of exponential concentration gradients

1976

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SynopsisContinuous exponential concentration gradients, accurate, precise, and reproducible to better than I% over a t least a 10"-104 concentration range, can be easily produced. In fact, it appears that these gradients may be substantially better than this. Such gradients have been used to generate a continuous ligand binding isotherm for the system Mg++ + Eriochrome Black T over a lo6 ligand concentration range. This continuous isotherm is shown to be sufficiently precise to provide a first derivative function r(y), which is precise to 0.4%. The moments of I'(y) were calculated and found to be extremely accurate. For example, the second moment about the mean was calculated to he 3.32 and 3.30 for two different experiments compared to a theoretical value of 3.29. The general application of these techniques to generate continuous ligand binding isotherms is discussed.

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Utilization of Automation for Studies of Enzyme Kinetics

Cited by 16 publications

References 34 publications

Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis

Clotrimazole Preferentially Inhibits Human Breast Cancer Cell Proliferation, Viability and Glycolysis

Sodium carbonate versus borate buffer for lactase quenching, laboratory work

Generation of continuous ligand–macromolecule binding isotherms. Use of exponential concentration gradients

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