A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
| Autor: | Rebecca F Stremel, Gavin Rumbaugh, James R. Sellers, Courtney A. Miller, László Radnai |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
General Chemical Engineering ATPase Nicotinamide adenine dinucleotide General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Adenosine Triphosphate ATP hydrolysis Lactate dehydrogenase Pyruvic Acid Animals Lactic Acid Enzyme Inhibitors chemistry.chemical_classification Adenosine Triphosphatases Myosin Type II General Immunology and Microbiology biology General Neuroscience Hydrolysis Metabolism NAD High-Throughput Screening Assays 030104 developmental biology Enzyme chemistry Biochemistry 030220 oncology & carcinogenesis biology.protein Adenosine triphosphate Oxidation-Reduction Pyruvate kinase |
| Zdroj: | Journal of visualized experiments : JoVE. (150) |
| ISSN: | 1940-087X |
| Popis: | ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes). |
| Databáze: | OpenAIRE |
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