Mitochondrial dehydrogenase, activation

After the addition of 30 pL of TetraColar One reagent, 300 pL of media mentioned above are incubated under 5% C02-air at 37°C for 2h. The optical density at 450 nm of the media containing the water-soluble tetrazolium salt produced by the enzyme reaction is read, and the cell number in cultures is measured by the modification (WST-8 method) of the MTT assay for mitochondrial dehydrogenase activity in viable cells [27]. 

Where two enzymes compete for the same substrate, we expect to see some form of metabolic control and in this case the concentrations of NADH and acetyl-CoA are the key controlling factors (Figure 6.44). When glucose is not available as a fuel, metabolism switches to 3- oxidation of fatty acids, which generates more than sufficient quantities of both NADH and acetyl-CoA to drive the TCA cycle and to maintain oxidative phosphorylation. Pyruvate dehydrogenase activity is suppressed and pyruvate carboxylase is stimulated by ATP, NADH and acetyl-CoA (strictly speaking by low mitochondrial ratios of ADP/ATP, NAD+/NADH and coenzyme A/acetyl-CoA), so... 

Hoshi, M. Takashima, A. Noguchi, K. Murayama, M. Sato, M. Kondo, S. Saitoh, Y. Ishiguro, K. Hoshino, T. Imahori, K. Regulation of mitochondrial pyruvate dehydrogenase activity by r protein kinase I/glycogen synthase kinase 3/1 in brain. Proc. Natl. Acad. Sci. USA, 93, 2719-2723 (1996)... 

An effect secondary to the activation of enzymes by increased calcium levels can be increased production of reactive oxygen and nitrogen species. Thus, activation of mitochondrial dehydrogenases increases NADH production and electron transport, yet increased calcium uncouples ATP synthesis, and the excess electron generates superoxide. Calcium also activates nitric oxide synthetase. 

Malate dehydrogenase activity would be expected in intact mitochondria, but not in SMPs. The activity of this enzyme in mitochondrial fractions may be estimated by a spectrophotometric assay. Oxaloacetate and NADH are incubated, and the disappearance of NADH is monitored at 340 nm. NAD+ does not have strong absorption at this wavelength. Note that the reverse reaction is studied because the reaction as shown above is very unfavorable in thermodynamic terms ACT = +30 kj/mol). 

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

LeFlir M, Dubach U. Peroxisomal and mitochondrial b-oxidation in the rat kidney. Distribution of fatty acyl coenzyme A oxidase and 3-hydroxyacyl-coenzyme A dehydrogenase activities along the nephron. J Flistochem Cytochem 1982 30 441-444. 

The most commonly used approaches are the neutral red assay (cell viability and membrane damage), the Lowry (labeled proline), Coomassie blue, and Kenacid blue assays (cell proliferation and total cell protein), the MTT or tetrazolium assay (mitochondrial function), and the intracellular lactate dehydrogenase activity test (cell lysis). 

The MTT assay is based on the reduction of the yellow-colored MTT by mitochondrial dehydrogenase of metabolically active cells to a blue formazan that can be measured spectrophotometrically. 

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