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Molecular Cloning of Renal Cysteine Conjugate β-Lyase.

Schofield, Miles Alexander. (1993) Molecular Cloning of Renal Cysteine Conjugate β-Lyase. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

The mercapturic acid pathway is an important detoxication mechanism in mammalian cells/tissues. However, work over the last decade has shown that several halogenated alkenes, once processed via this pathway, become substrates for the enzyme cysteine conjugate β-lyase. This latter enzyme cleaves these conjugates to ultimately yield potent nephrotoxins. The factors controlling the β-lyase enzyme's cellular distribution and substrate specificity have, however, yet to be elucidated. To this end, a cDNA for cysteine conjugate β-lyase has been isolated from a rat kidney cDNA library using immunological screening. The cDNA was sequenced and found to be previously unreported. Northern blot analysis of rat kidney and liver RNA showed the cDNA to be highly specific for a single mRNA, of 1.9kb in length, in the kidney which was in agreement with the expected size for a 48kD protein. This cDNA was used to investigate the tissue distribution of rat kidney β-lyase. Northern blot analysis of tissue RNA was in close agreement with the Western blot finding that the kidney β-lyase was of a structurally different form when compared to extrarenal forms of the enzyme. The cDNA was also used to examine the induction/reduction of rat kidney β-lyase activity by pretreatment of rats with the β-lyase substrate precursor NAc-PCBD. It was demonstrated that a non-toxic dose of 3mg/kg NAc-PCBD caused a significant increase in kidney cytosolic β-lyase as assessed by enzyme activity, enzyme protein levels and corresponding mRNA levels. In contrast, a toxic 10mg/kg dose caused a significant decrease in these latter parameters. Further cDNAs were isolated by immunological screening of a separate cDNA library but failed to be identified as corresponding to β-lyase. The possible reasons for this and the significance of the isolation and characterisation of the original cDNA are discussed at length.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Schofield, Miles Alexander.
Date : 1993
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1993.
Depositing User : EPrints Services
Date Deposited : 14 May 2020 14:03
Last Modified : 14 May 2020 14:08
URI: http://epubs.surrey.ac.uk/id/eprint/856449

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