Plant endogenous beta-glucuronidase activity: how to avoid interference with the use of the E. coli beta-glucuronidase as a reporter gene in transgenic plants.
We have detected a plant beta-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric beta-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45 degrees C. It is relatively resistant to proteases and insensitive to -SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by the Escherichia coli beta-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay the E. coli beta-glucuronidase in transgenic plants without interference from the endogenous plant activity.
Alwen
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Institute of Microbiology and Genetics, University of Vienna, Austria.
Benito Moreno
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Vicente
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Heberle-Bors
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D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005821:Genetic Techniques; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism*; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D013347:Subcellular Fractions / Q000201:enzymology; D014026:Nicotiana / Q000201:enzymology*
D005966:Glucuronidase