Drake, Beryl2008-09-022022-11-022008-09-022022-11-0219821982https://ir.wgtn.ac.nz/handle/123456789/29062The purification of GSH S-transferases from Wiseana larvae is described. The purification procedure developed was DE-32 cellulose chromatography, affinity chromatography on GSH-BSP coupled to epichlorohydrin-activated sepharose 4B, CM-cellulose chromatography and preparative isoelectric focusing. Glutathione S-transferase IVb was purified to apparent homogeneity as judged by disc electrophoresis, SDS-electrophoresis and isoelectric focusing. The protein had an isoelectric point of 7.91. A partially pure enzyme, GSH S-transferase I, on preparative isoelectric focusing yielded two enzyme fractions Ia and Ib. The most active fraction, Ib, had an isoelectric point of 6.62. The molecular weight of GSH S-transferases I and IV was estimated to be 40,000. SDS-electrophoresis revealed a single band of molecular weight of 25,500 for GSH S-transferase I and 22,900 for GSH, S-transferase IV, indicating that the enzymes are each composed of two subunits of approximately equal size. The optimum pH for enzyme activity was 8.0 to 8.5 for both GSH.S-transferase I and IV. Both enzymes are active for CDNB, DCNB, ethacrynic acid and iodomethane conjugation but were inactive for 4-nitropyridine-N-oxide, 1, 2-epoxy-3-(P-nitrophenoxy)propane, BSP and P-nitrobenzyl chloride conjugation. GSH S-transferase IV has no detectable activity with the insecticides methyl parathion and diazinon. Despite similar physical properties GSH S-transferases I and IV are not immunologically related. GSH S-transferase IV was shown to have a hiqh content of the amino acids lysine, aspartic acid, glutamic acid and leucine. The kinetics of GSH S-transferase IV are complex and resemble those reported for GSH S-transferase A from rat liver, except that the enzyme activity is inhibited by the product, chloride ion. The localisation method utilising polyacrylamide-starch gels and the substrate iodomethane was developed for the detection of GSH S-transferase activity.pdfen-NZBiochemistryWiseana larvaeEnzymesGlutathione S-transferasePestsInjurious and beneficial insectsText