In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile.

Abstract

We have previously reported that when garter snakes. Thamnophis sirtalis parietalis, a freeze tolerant species, were exposed to 5 h freezing at -2.5 degrees C organs showed increases in the activities of anti-oxidant enzymes, especially catalase in skeletal muscle. This was interpreted to be an adaptation to deal with the potentially injurious postischemic situation of thawing. The present work analyzes in vitro oxidative inactivation of a possible target of postischemic-induced free radical damage, the secondary anti-oxidant defense glutathione-S transferase, and the protective role of endogenous catalase. Approximately 50% of GST activity from snake muscle homogenates was lost within 2 min after addition of H2O2 plus Fe(II) (0.4-2 mM) in media containing azide whereas addition of iron alone resulted in no damaging effects. The opposing effects of dimethyl sulfoxide and EDTA in modifying this process strongly suggested the involvement of .OH radicals in the GST inactivation. A partial recovery of the activity was promoted by mercaptoethanol, indicating that sulphydryl groups oxidation participate in the mechanism of GST inactivation. Pre-incubation of the reaction media containing H2O2 caused protection of the GST activity only in the absence of azide, indicating that endogenous catalase modulates the extent of oxyradical damage. The protective pre-incubation effect was more efficacious when employing homogenates from lung and liver, organs that have higher catalase activities, as well as homogenates from freezing-exposed muscle (that show an 80% increase in catalase activity, compared with control). The protection against GST inactivation observed in muscle from frozen snakes demonstrates that increased anti-oxidant defenses during freezing exposure can be a key factor in controlling in vitro oxyradical damage. The implications for natural freeze tolerance are discussed.