Lange et al. (2002) showed that the hepatic level of total GSH increased in rainbow trout after 14 days’ exposure to Cd by about 1.5 times compared to the control, but after 28 days no significant changes were observed. Gil & Pla (2001) postulated that GSH could serve as a biomarker for a variety of xenobiotics. In order to gain a better understanding of the part played by GSH in protecting malic enzyme from cadmium toxicity, we studied how the GSH level would affect the inhibition of malic enzyme activity by cadmium. In the muscles of crustaceans this enzyme is involved in NADPH formation, which is important
in detoxification processes. The toxic effect of cadmium was tested in vitro by using the NADP-dependent malic http://www.selleckchem.com/products/ly2157299.html enzyme, activated by divalent cations, from shrimp abdominal muscles. Some of our results suggest that the presence of cellular
GSH reduces cadmium inhibition of NADP-dependent malic enzyme and in consequence protects this enzyme. Brown shrimps Crangon crangon 3–4 cm in length were caught in the Gulf of Gdańsk off Sobieszewo Island near the delta of the River Vistula in June and July and kept in aerated seawater. Malic enzyme (L-malate: NADP oxidoreductase (decarboxylating) E.C. 18.104.22.168) activity at all purification steps was tracked spectrophotometrically with a UV-VIS recording spectrophotometer by observing the appearance of NADPH at 340 nm and Selleck Quizartinib 25°C. The standard reaction mixture contained 50 mM Tris-HCl, pH 7.5, 0.5 mM NADP, 5 mM L-malate and 1 mM manganese chloride. Enzyme activities were calculated using E mM × 340−1 = 6.22 for NADPH in a 1 cm light-path quartz cell. Protein concentration was determined by Spector’s (1978)
method. Shrimp malic enzyme (ME) (L-Malate: NADP oxidoreductase (decarboxylating) EC 22.214.171.124) was isolated from the abdominal muscles of brown shrimps PRKACG C. crangon caught in the Gulf of Gdańsk and purified to the specific activity of 20 μmols min−1 mg−1 protein by the method described by Skorkowski & Storey (1987). Sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) was performed according to Laemmli’s method (1970), the marker being SDS-7B (Sigma-Aldrich). The samples were subjected to electrophoresis at 20 mA, 25°C for 2.5 h, and the gel was stained with Coomassie Brilliant Blue. The muscles of brown shrimps C. crangon (about 200 mg of the tissue) were homogenized in 1 ml buffer, pH 3.5 (H2O : ACN, 90 : 10 v : v, with 1 mM ammonium acetate). After centrifugation (800 g, 5 min) a 100 μl sample of the supernatant was obtained. The supernatant was adjusted with the buffer, pH 3.5, to a volume of 300 μl. Linearity was tested using five standards from 0.1 to 10 mg l−1 (0.1, 0.5, 1, 5, 10 mg l−1) for GSH. GSH was analysed on a ThermoQuest Finnigan LCQ Deca mass detector equipped with ESI interface (Finnigan, USA). A Kinetex C18 (100 × 4.6 mm, 2.