Studies on Glutathione S-Transferases from the Australian Sheep Blowfly (Lucilia Cuprina)

Abstract

Glutathione S-transferases (GSTs) are a family of enzymes with a well established function of detoxification of a wide range of reactive and toxic compounds. In humans these enzymes have been implicated in development of resistance to chemotherapeutic drugs and in insects they are protective by conferring resistance against insecticides. Recently, it has been shown that GSTs have additional functions as transport proteins and as a defence mechanism against oxidative stress. This project focuses on the GSTs from the Australian sheep blowfly, Lucilia cuprina with emphasis on the specific Sigma and Delta (originally described as Theta) class of enzymes. Though there is not much information on the Sigma class of GSTs, they are said to be involved particularly in the repair of oxidative damage to cellular components. The Delta class of GSTs are shown to be involved in development of resistance in insects against insecticides. Sigma and Delta GSTs were isolated from Lucilia cuprina using GSH affinity chromatography. Chromatofocusing was then employed to separate the two enzyme classes. The purified proteins were characterised using 1D and 2D gel electrophoresis. MALDI-TOF mass spectrometry helped identify the purified proteins as GSTs and also proteins co-isolated by the column. The next phase of the project focused on determining the inhibitory properties of native plant extracts towards the purified Sigma and Delta GSTs and also towards crude enzyme. BSP-GSH affinity chromatography was employed to isolate proteins other than those purified by the GSH affinity column. Proteins isolated were visualised by gel electrophoresis and identified by mass spectrometry. To try and learn more about the Sigma and Delta GSTs, an attempt was made to isolate then from the developmental larval and pupal stages of Lucilia cuprina. Interestingly, the Lucilia larvae showed complete absence of Sigma GSTs, while a trace of developing Sigma was found in the pupal stages. These expression differences are in line with the view that the Sigma class of GSTs has a function to play in the adult flies other than that of general detoxification.