Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions.

Abstract

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.

Redox Balance Mechanisms in Schistosoma mansoni Rely on Peroxiredoxins and Albumin and Implicate Peroxiredoxins as Novel Drug Targets

“…Protein concentrations were determined using the Bradford assay according to the manufacturer's protocol (Bio-Rad). Lactate dehydrogenase and succinate dehydrogenase activities (27,28) were used as controls for cytoplasmic and mitochondrial fractions, and histone and sarco/endoplasmic reticulum calcium ATPase proteins, detected by Western blot, served as controls for the nuclear and microsomal fractions, respectively.…”

Preeclamptic placental stress and over expression of mitochondrial HSP70

“…Succinate dehydrogenase activity was assayed by the method of Green and Narahara (17). The spectrophotometric cuvette contained the following reagents in a total volume of 4 mL: 1.0 mL of phosphate buffer, 0.1 mL of EDTA, 0.1 mL of BSA, 0.1 mL of KCN, 0.3 mL of sodium succinate, 0.2 mL of potassium ferrocyanide and 2 mL of distilled water.…”

Peroxisome Proliferator-activated Receptor γ Coactivator 1α or 1β Overexpression Inhibits Muscle Protein Degradation, Induction of Ubiquitin Ligases, and Disuse Atrophy

“…Then, total protein was measured using Coomassie Plus (Pierce). Citrate synthase activity was determined spectrophotometrically at 30°C as done by Srere (35), and succinate dehydrogenase activity was measured at 37°C as done by Green and Narahara (36).…”