No abstract

Purified juvenile catfish (Clarias gariepinus) glutathione transferase (cgGST) was denatured in vitro and renatured in the absence and presence of different concentrations of endogenous or xenobiotic model substrates. Protein transitions during unfolding and refolding were monitored by activity measurement as well as changes in protein conformation using UV difference spectra at 230 nm. Gdn-HCl at 0.22 M caused 50 % inactivation of the enzyme and at 1.1 M, the enzyme was completely unfolded. Refolding of cgGST main isozyme was not completely reversible at higher concentrations of Gdn-HCl and is dependent on protein concentration. An enzyme concentration of 30 μg/ml yielded 40 % percentage residual activity in the presence of glutathione (GSH), regardless of the concentration that was present as opposed to 30 % obtained in its absence. The xenobiotic model substrate, lindane, appears to have no effect on the refolding of the enzyme. In summary, our results show that GSH assists in the refolding of cgGST in a concentration-independent manner and may be involved in the same function in vivo whereas the xenobiotic model substrate does not.

Catfish are hardy in nature and it is not known whether the presence of efficient detoxication enzymes is partly responsible for this trait. To investigate this, we have assessed induction of glutathione transferase (GST) in 10-week-old juvenile catfish (Clarias gariepinus) exposed to graded concentrations of lindane, an organochlorine insecticide, and characterised the purified enzyme from groups having the highest and statistically significant induction. Some of the unique properties observed for the purified enzyme are a high K m (1.7270.21 mM) for the electrophilic model substrate, 1-chloro-2,4-dinitrobenzene (CDNB) and a very low catalytic rate (V max =0.13070.010 units/mg protein). The k cat /K m being 55.470.2 M À 1 s À 1 . The enzyme is present in high concentration in the organism, the main isoform accounts for about 5.6% of the total soluble protein, probably to compensate for the observed kinetic imperfection. Since these properties are generally not known for a detoxication enzyme, we suggest that they may form part of the organism's own adaptation to its polluted environment.

Th e varying status of glutathione transferases (GSTs) in water snail, Bulinus globosus , an intermediate host of disease-causing Schistosoma haematobium (Bilharz 1852) has been investigated. Th e expression of GST isoenzymes in the water snail appears seasonal with about three isoenzymes appearing during raining season, when the organism is active, which may reduce to a single peak of one isoenzyme during aestivation, when the organism is inactive. GST isoenzyme is present in high concentration in all the tissues investigated namely: haemolymph, foot muscle and hepatopancreas with specifi c activities of 0.006 ± 0.002, 0.45 ± 0.021 and 1.33 ± 0.103 units/mg protein respectively for 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. With this substrate, the specifi c activity of GST from the hepatopancreas appears higher than the specifi c activities that have been previously reported for GSTs from molluscs. Partial purifi cation of the isoenzymes using Tris acrylic acid-based resins enabled us to observe that GST appears to be the major protein in the hepatopancreas of this organism. We also found indications for the presence of an endogenous GST inhibitor in the cytosol, whose function is yet unknown. All the traditional GST inhibitors such as cibacron blue, hematin, bromosulfophthalein and S-hexylglutathione were able to inhibit the isoenzymes eff ectively, with cibacron blue being the most potent. Th e isoenzymes however have narrow substrate specifi city. We conclude that diff erent isoenzymes of GST are expressed in the same class of molluscs, even when they belong to the same genus or species, and that the expression may depend on whether the snails are on aestivation or not.