Studies on Glutathione S-Transferases and Glutathione-Requiring Dehalogenases From Mammalian Liver

Abstract

Part 1. The involvement of GSH-dependent enzymes in the metabolism of fluorinated compounds. The GSH-requiring dehalogenase from rat liver does not appear to be one of the previously characterised GSTs and has no activity towards the model substrates of the GSTs. However, the enzymes do have some properties in common. The defluorinating activity is cytosolic and has an absolute requirement for glutathione. The dehalogenases from rat, mouse and possum liver appear to have a relative molecular mass of approximately 40000. The dehalogenase is inhibited by inorganic anions in what appears to be a similar manner to the GSTs. In mouse and possum liver at least two forms of the dehalogenase are present, with isoelectric points above and below pH8. Fluoroacetate(Compound 1080) was the only substrate tested that showed appreciable catalysed conjugation with glutathione. The dehalogenase was present in all the mammals tested. Insects exhibited no activity, and activity was minimal in fish. Part 2. Types of affinity matrix for the purification of the glutathione S-transferases and glutathione-requiring dehalogenases Glutathione and S-hexyl glutathione were attached directly to epichlorhydrin-activated Sepharose such that there was no spacer group between the ligand and the matrix. The performance of these matrices for the purification of the major rat GSTs was compared with a matrix that used a GSH-BSP conjugate ligand, also attached to epichlorhydrin-activated Sepharose. Use of the GSH-matrix resulted in a 9l% recovery of the GST activity witha 19.7 fold purification. With the GSH-BSP conjugate matrix, 98% of the activity was recovered witha 12.7 fold purification. The S-hexyl GSH matrix did not bind GST 1-1. The recovery was 50% witha 8.9 fold purification. The rat GSTs did not bind to affinity matrices made with epoxy-activated cellulose supports even though the matrices were successfully substituted with GSH and GSH-BSP conjugate ligands. The glutathione-requiring dehalogenating activity bound only to the GSH-BSP conjugate ligand attached to epichlorhydrin-activated Sepharose. Part 3. Interactions between the glutathione transferase active site and inorganic anions The rat glutathione S-transferases 1-1, 3-3, 3-4, and 4-4 were found to be inhibited in a noncompetitive manner by inorganic anions in the order: F-< Cl- < Br- < NO3- < I- < ClO4- < SCN- with only ClO4- and SCN- changing places for some isoenzymes. The rat liver dehalogenase activity was also inhibited by these anions in the same order. The order appears to be dependent on the enthalpy of hydration of the anions. A model of GST catalysed conjugation of GSH and CDNB is proposed. In addition to base assisted deprotonation of glutathione, it is proposed that a positively charged group in the GST active site assists in the leaving of the chloride ion from CDNB. Abbreviations: BSP – sulphobromophthalein CDNB – 1-chloro 2, 4-dinitrobenzene GSH – glutathione (reduced) GST – glutathione S-transferase I would like to extend my special thanks to my supervisor, Dr Alan Clark, for his guidance and encouragement throughout this project. I would also like to thank David Hall, Barry and Adrienne Marshall, Bruce Rae, and Richard Moore. I have been supported for the greater part of this work by a medical Research Council of New Zealand Post-graduate Scholarship. Funding for chemicals and equipment was provided by a grant from the Wellington Medical Research Foundation to Dr A. G. Clark.