Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. A methodological study.

Abstract

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 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees 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 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 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.