Abstract
A major route of detoxification for many halogenated xenobiotics is by conjugation to the tripeptide glutathione. Subsequent metabolic processing of the glutathione conjugate yields the corresponding cysteine conjugate of the xenobiotic, which may then serve as a substrate for enzymes which express beta-lyase activity. Kidney cysteine conjugate beta-lyase [glutamine transaminase K (GTK), kynurenine aminotransferase (KAT)] is an enzyme which metabolises cysteine conjugates of certain halogenated alkenes and alkanes to form reactive metabolites. Such metabolites produce nephrotoxicity in experimental animals, neurotoxicity in rabbits, and may produce subtle kidney changes and neurotoxicity in man. Although the role of kidney cysteine conjugate beta-lyase in the production of nephrotoxic thiols from cysteine conjugates of xenobiotics has been well established, the factors controlling the cellular distribution and substrate specificity of the enzyme have yet to be elucidated. Additionally, the recent finding by other researchers that the rat brain beta-lyase enzyme also possesses KAT activity raises the possibility that it may play a role in neurotransmission via modulation of the NMDA receptor. As an approach to understanding the species specific differences in halogenated alkene nephrotoxicity, the full-length cDNAs for both rat and human kidney cysteine conjugate beta-lyase have been isolated using a combination of hybridisation screening, 5' RACE and RT-PCR technologies. The cDNAs were sequenced and their identities confirmed by deduced molecular weight, deduced amino acid composition, the presence of a consensus pyridoxal phosphate binding site in their deduced amino acid sequences, and expression of the cDNAs in tissue culture cells. The rat and human cDNAs coded for proteins of 47.8 kDa and 47.9 kDa respectively, consistent with the published values for the purified beta-lyase proteins. Cytosolic extracts of COS-1 cells, transfected with the rat and human cDNAs demonstrated significant increases in both beta-lyase and GTK activity, with approximately similar Km values for the rat and human enzymes. Western blot analysis using the purified antibody to rat kidney cytosolic beta-lyase, was performed on cytosolic extract from COS-1 cells transfected with the rat cDNA and demonstrated a protein band identical in its position to that of authentic rat kidney beta-lyase. Comparison of the human deduced amino acid sequence with that of the rat enzyme indicated an 82% overall similarity, with 90% similarity either side of the pyridoxal phosphate binding site (residues 187-289), many of the changes being conservative in nature. Preliminary mapping of the gene for human cysteine conjugate beta-lyase, using PCR analysis of genomic DNA from human-rodent hybrid cells, indicated that it is located on human chromosome 9. The use of the rat and human beta-lyase cDNAs for the elucidation of the mechanism of action, substrate specificity, localisation and potential toxicity of these enzymes is discussed in this thesis. The work presented here, has given rise to a number of poster presentations, oral communications and refereed publications (see appendix II).