Adaptation of the tetrazolium reduction test for the measurement of the electron transport system (ETS) activity during embryonic development of medaka

Abstract

The quantitative electron transport system (ETS)‐assay based on tetrazolium reduction has been adapted for determining the terminal ETS activity during embryonic development of the subtropical teleost fish Oryzias latipes, medaka. Homogenization with a glass potter for 1–2 min was required for the complete extraction of the ETS. Additional sonication and centrifugation had a degradatory effect on the ETS activity. The main substrate of the ETS of fish embryos was NADH. NADPH also donated electrons for the ETS but with much less intensity. The impacts of the NADH and the NADPH on the enzyme activity was not additive. Succinate was ineffective as a substrate for the ETS. NADH (1.7 mM) and NADPH (0.25 mM) in combination with 0.8 mM of the artificial electron acceptor, 2‐(p‐iodophenyl)‐3‐(p‐nitrophenyl)‐5‐phenyl tetrazolium chloride (INT), ensured a Vmax for the ETS if the reaction mixture contained 400 μg wet weight egg ml−1 of cell‐free homogenate. The pH‐optimum of the ETS was between pH 8.0 and 8.6. The enzyme reaction at 24°C was linear during 40 min incubation. The ETS activity increased exponentially during embryonic development. The assay could be a useful tool for detecting the effect of pollutants on the development of the respiratory enzyme system in fish during embryogenesis.