Oxidative Stress Poster Session
Materials and Methods
All materials were purchased from Sigma Chemical Company and were of the highest purity available, unless otherwise indicated. Toads were live-shipped from Arizona and were treated as described in Grundy and Storey (1998). Briefly, animals were washed and treated with a mild solution of tetracycline. Upon observation that the toads had recently fed, 1/2 of the toads were killed by pithing and their tissues rapidly excised and frozen in liquid nitrogen, then removed for storage at -70 ° C until use. The remaining toads were placed on containers of sterile soil covered with a wire grille. They rapidly burrowed and estivated for two months in an incubator at 15 ± 2 ° C, then were killed and their tissues stored as for awake toads.
GST activity was measured using the method of Habig and Jakoby (1981). The assay measures the formation of a conjugate between GSH and 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm (extinction coefficient = 9.6 cm-1 mM-1) in 50 mM potassium phosphate buffer, pH 6.5 with 1 mM EDTA. The reaction was started by the addition of GST. One unit of activity was the amount of GST required to produce 1 micromole of conjugate per minute.
Peroxidase activity (both selenium-dependent and selenium-independent) was measured by the method of Ahmad and Pardini (1988). Each 1 mL coupled-enzyme assay contained 50 mM potassium phosphate, pH 7.0, 1 mM EDTA, 2 mM sodium azide, 0.2 mM NADPH, 5 units of Glutathione Reductase (Sigma), 5 mM GSH (1 mM for frog liver) and 1.2 mM cumene hydroperoxide. Reaction was started by the addition of GST. One unit of activity was the amount of GST which resulted in the reduction of 1 nanomole of NADPH per minute (extinction coefficient = 6.22 cm-1 mM-1).
GST was purified from frog (Rana pipiens) liver and leg muscle and from toad (Scaphiopus couchii) liver and leg muscle in the following manner:
Substrate affinity constants (Km) of GSH and CDNB were determined for all GSTs along with half-maximal inhibition (I-50) values. Vmax (optimal) conditions were: 5 mM GSH for toad liver, 2 mM GSH for all other tissues; 2 mM CDNB for toad liver and frog muscle, and 1 mM for all other tissues. Temperature dependence of GST activity was assessed over the range from 2-3 ° C up to 23 ° C for frog muscle GST and 40-45 ° C for all other samples. Activity was measured under Vmax conditions; temperature effects were analyzed using an Arrhenius plot of log V versus 1/ ° K.
In a separate experiment, supernatant from liver of awake or estivated toads was separated by isoelectric focusing using an LKB 8101 column in a sucrose gradient, with ampholines of pH range from 3.5 to 10.
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|Grundy, J.E.; Storey, K.B.; (1998). Purification and Kinetic Properties of Glutathione-S-transferase from Liver and Skeletal Muscle of the Spadefoot Toad, Scaphiopus couchii: Influence of Estivation.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/oxidative/grundy0446/index.html|
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