Oxidative Stress Poster Session



Materials & Methods


Discussion & Conclusion



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Purification and Kinetic Properties of Glutathione-S-transferase from Liver and Skeletal Muscle of the Spadefoot Toad, Scaphiopus couchii: Influence of Estivation.

Contact Person: Kenneth B Storey (kenneth_storey@carleton.ca)

Discussion and Conclusion

Reduced specific activity of GST in estivated tissues indicates either: reduced GST expression during estivation, catabolism of the enzyme, modulation of GST with allosteric effectors, post-translational modification of the protein itself, resulting in reduced activity, or expression of isozymes of GST with different specific activity, or a combination of the above (Storey, 1992). The first two circumstances would yield the identical enzyme- kinetic properties would not be altered. Most low molecular weight allosteric effectors would be diluted out during homogenization and desalting. Comparison of the kinetics of the enzyme from awake or estivated animals thus permits determination of whether the enzyme is different during estivation, or present in lower amounts.

  • The reasons for lower GST activity in toad liver and muscle during estivation were different and tissue-specific, as shown by kinetic analysis and characterization of the purified enzymes.

    Toad Leg Muscle

    GST from leg muscle of awake toads was similar in every respect kinetically to the enzyme isolated from leg muscle of estivated toads. Specific activity and subunit molecular weight were also similar. The enzymes were concluded to be the same and were pooled for further tests. Lower GST activity in estivated muscle may thus be due to decreased enzyme levels.

    Toad Liver

    The final specific activity was different between the GSTs isolated for awake and estivated toads, and further analysis revealed differences in pI and kinetic parameters. This is consistent with post-translational modification or production of a different GST during estivation. Neither toad liver isozyme was identical to the GST from toad muscle, indicating tissue-specific GST isozyme distribution in toads.

  • All forms of toad GST that were isolated had characteristics of the Mu class of GST isozymes. Specifically, peroxidase activity, a hallmark of Alpha class GST, was low to undetectable in purified toad GSTs. Toad liver GSTs showed similar isoelectric points to those of Mu (near-neutral) class. Both liver and muscle GSTs showed kinetic inhibition profiles with cibacron blue, rose bengal and s-hexylglutathione which were similar to Mu class inhibition.

    GST from Frog Liver and Muscle

    Two forms of GST were isolated from frog liver: one form (GST A) showed activity with both CDNB and with cumene hydroperoxide, indicating that GST A was likely of the Alpha class. This was strengthened by the results of inhibition with cibacron blue, which was similar to human Alpha class GST. Frog liver GST B, and frog leg muscle GST showed inhibition characteristics similar to Mu class isozymes.

    Comparison of Frog and Toad GSTs

    Frog liver GST B and toad liver GSTs had higher specific activity than the muscle isozymes and had similar activation energies. Purified muscle GSTs from frog and toad showed similar kinetic constants, specific activity of the purified enzyme, and activation energy. With the exception of GST from frog muscle, frog and toad GST subunits were of comparable size. This may indicate the existence of common GST isoforms in amphibian liver and muscle.


    GST was isolated from liver and muscle of Scaphiopus couchii, to examine the cause of reduced activity in these tissues during estivation. In leg muscle, the enzyme levels were reduced but the enzyme itself was unchanged during estivation, whereas kinetic data showed that the enzyme in liver of estivated toads was different from that from awake toads. Thus, changes in GST activity during estivation in spadefoot toad liver and leg muscle are tissue-specific, and in liver, may include transcriptional or post-translational changes.

    A comparison of GSTs from S. couchii to enzymes from leopard frogs, Rana pipiens, showed similarities between GSTs from the same tissue, with the exception of GST A from frog liver, which had different kinetic properties, substrate acceptance and activation energy from the other GSTs. Frog and toad GSTs showed inhibition characteristics of the Mu class of GST isozymes, except for GST A from frog liver, which showed similarity to Alpha class GSTs.

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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
    © 1998 Author(s) Hold Copyright