Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

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:
Frozen tissue was homogenized 1:5 in 5 mM potassium phosphate, pH 6.0 containing 1 mM EDTA (Buffer A). The homogenate was centrifuged at ca. 28,600 x g for 20 minutes at 4 ° C in a refrigerated ultracentrifuge. Supernatant was desalted by elution through a small column containing Sephadex G-25 equilibrated in Buffer A. An aliquot of desalted supernatant was loaded onto a hydroxylapatite (Bio-Rad) column equilibrated in Buffer A. The column was washed to remove non-adsorbent protein and GST was eluted with a linear gradient of 5-500 mM potassium phosphate, pH 6.0, containing 1 mM EDTA. Enzyme activity was measured and peak fractions were pooled and desalted by elution through G-25 or by dialysis against Buffer A. Desalted eluant was then applied to an S-hexylglutathione agarose affinity column, previously equilibrated in Buffer A. The column was washed with Buffer A, then with 5 mM potassium phosphate, 1 mM EDTA at pH 9.0, then GST activity was eluted with three sequential washes of 5 mM GSH in the pH 9 buffer. Activity with CDNB was monitored at all stages of the purification; activity with cumene hydroperoxide was tested in final preparations of the purified enzyme. Samples were retained from all steps of the purification for measurement of protein content. Final samples were run on SDS-PAGE and the gel was stained with silver to test for purity.

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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