|
(22-43) Inhibition of Seed Protein Catabolism and of Shott Protein Anabolism After Exposure to Heavy Metals. Gianazza, Elisabetta1, Tome', Franca1, Di Fabio, Tiziana1, Regondi, Simona1, Wait, Robin2, Agradi, Elisabetta*,1, 1 Universita' degli Studi di Milano, Milano, Italia2 Imperial College of Science, Technology and Medicine, London, U.K. ABSTRACT- Nasturtim officinale seeds were germinated in the presence of varying concentrations of different inorganic pollutants (copper, cadmium, chromium, zinc, iron); growth of the shoots was inhibited in a dose-dependent way. Proteins were extracted from whole plantlets by homogeneization in a SDS-containing buffer, at a 4:1 ratio to fresh weight. The protein extracts were analyzed by electrophoresis on a 4-20 % polyacrylamide gradient and stained patterns were quantitated by densitometry. In treated samples several bands were found at a much higher concentration than in controls. Most of the relevant components were 30, 25 and 10 kDa in size. To identify the proteins affected by toxic exposure, N. officinale extracts were resolved by 2DE (two-dimensional electrophoresis); tens of spots were recognised that migrated as rows of components with similar mass and varying isoelectric point. Spots of interest were excised and digested in-gel with trypsin; the resulting peptide fragments were analysed by ESI-MS/MS (mass spectrometry by electrospray ionisation with collision fragmentation of the daughter ions). No genomic information is available for N.officinale; however, partial sequencing by ESI-MS/MS allowed to define already characterised proteins with closer structural relationship to the target. For most spots homology was with storage proteins from Cruciferae seeds, either Arabidopsis thaliana or Brassica napus. This finding allows to describe the inhibition of plantlet development by heavy metals as a blockade of the degradation of seed proteins at the same time and to the same extent as a blockade of the de novo biosynthesis of shoot proteins. Research sponsored by EC (CITYFISH). Key words: proteomics, heavy metals, two-dimensional electrophoresis, mass spectrometry |
