Lao, Si-houy (2003) Detoxification of 3,4-dichloroaniline in soybean by n-malonylation. Doctoral thesis, Durham University.
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3,4-Dichloroaniline (DCA), the degradation product of certain herbicides, is not readily degraded by micro-organisms and, due to its persistence in the environment, is considered to be a reference xenobiotic. Here, the metabolic fate of [UL-(^14)C]-3,4-DCA was investigated in soybean (Glycine max var. Chapman) plants over a 48 h period following treatment via the root media. DCA was rapidly taken up and metabolised to 7V-malonyl-DCA. Synthesis occurred in the roots and the conjugate was largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once exported, the DCA metabolites present in the medium were not readily taken up by soybean roots. Conjugation and export of DCA therefore constitute an effective detoxification mechanism for the plant. A radiometric assay for DCA-N-malonyltransferase (E.C.22.214.171.124; DCA-N-MT) was developed and used to follow DCA-N-MT activity through a four-step protocol, in which DCA-N-MT was purified 400-fold from soybean roots. SDS- PAGE analysis and gel filtration chromatography suggested that DCA-N-MT is a 52 ± 2 kDa protein. Following treatments with 100 μM DCA for 24 h, DCA-N-MT activity in soybean roots increased from 44.6 ± 8.1 nkatg(^1) to 104 ± 4.9 nkatg(^1) but did not vary significantly in suspension-cultured cells (332.9 ± 38.9 nkat.g(^1)). Kinetic studies suggested that this increase in activity could be due to de novo protein synthesis. Partially-purified DCA-N-MT was therefore subjected to differential gel electrophoresis (DiGE) analysis to identify proteins which increased in abundance in response to DCA pre-treatment. Although a clear candidate for DCA-N-MT was not detected, five differentially expressed proteins were identified by mass spectrometry.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||09 Sep 2011 10:00|