Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues
| Autor: | Remco J. Molenaar, Mohammed Khurshed, Cornelis J.F. Van Noorden, Vashendriya V V Hira |
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| Přispěvatelé: | Cell Biology and Histology, Graduate School, Medical Biology, AGEM - Endocrinology, metabolism and nutrition, AGEM - Digestive immunity, CCA - Cancer biology and immunology |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
General Chemical Engineering Dehydrogenase Cofactor General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Issue 135 NAD+ NADPH Humans Immunology and Infection chemistry.chemical_classification biology General Immunology and Microbiology Chemistry Histocytochemistry General Neuroscience activity Subcellular localization Immunohistochemistry enzyme 030104 developmental biology Enzyme Biochemistry histochemistry cytochemistry dehydrogenase NADP+ NADH biology.protein Cytochemistry NAD+ kinase Formazan Oxidoreductases Quantitative analysis (chemistry) 030217 neurology & neurosurgery Quantitative |
| Zdroj: | Journal of Visualized Experiments : JoVE Journal of visualized experiments, 2018(135):e56843. MYJoVE Corporation |
| ISSN: | 1940-087X |
| Popis: | Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis. |
| Databáze: | OpenAIRE |
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