Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells

Summary

Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at −20° C), coating of cells with CoQ₁₀ (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37° C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6. PMS as an intermediate electron carrier was found inferior to exogenous CoQ₁₀. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37° C. The apparent K _m values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I _0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.