Monochiorobimane (BmCl), a non-fluorescent cell-permeant compound that reacts with glutathione to yield a strong hlue fluorescent conjugate bimane-glutathione (Bm-SG), was used to trace the glutathione-dependent detoxiflcation of xenobiotics in plant cells and protoplasts. In BmCl-labelled cells and protoplasts, fluorescence developed rapidly and was quickly concentrated in the vacuole. The rate of fluorescence development was dependent on the concentration of BmCl and the only metabolite formed was the conjugate Bm-SG. The formation of Bm-SG was correlated with a decrease in the amount of intracellular GSH. Compounds which reduced the intracellular levels of GSH severely reduced the formation of Bm-SG. Bm-SG was shown to be transported into isolated vacuoles by an ATP-dependent vanadate-sensitive mechanism. Kinetic analysis of cellular Bm-SG formation implicated both spontaneous conjugation and enzyme catalysis. Our results demonstrate a cellular pathway for xenobiotic detoxification in plants, starting with conjugation to glutathione in the cytoplasm, followed by the transport of the conjugates into the vacuole. This pathway is used to counter the toxic effects of some herbicides and environmental pollutants and overlaps with or parallels the pathway used for the biosynthesis of anthoeyanins.
Abstract. Polyclonal antiserum and monoclonal antibodies raised to a purified cutinase from Fusarium solani f. sp. pisi have been used to identify an active cutinase in the pollen of Brassica napus. These antibodies recognized a polypeptide with an estimated molecular weight of 22-kDa -a molecular weight indentical to that of the Fusarium cutinase -and localized this polypeptide to the intine of the pollen wall. Enzyme assays on the renatured 22-kDa polypeptide after electroelution from a preparative SDS-PAGE gel revealed the polypeptide to be an enzyme capable of cataiysing the hydrolysis of tritiated apple cutin and the synthetic substrate p-nitrophenyl butyrate. The molecular weight, immunological properties and substrate specificity of the Brassica cutinase suggest that this enzyme resembles more closely fungal cutinases than it does the cutinase from the pollen of Nasturtium (Tropaeolum majus) the only angiosperm cutinase so far characterized (Maiti et al., 1979, Arch. Biochem. Biophys. 196, 412-423). These differences between the pollen cutinases from two members of the Dicotyledoneae are unexpected and predict a diversity of this class of pollen enzyme within the angiosperms.
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