Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance |
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Authors: | Benjamín Frydman Rosalía B Frydman Carlos de Los Santos Daniel Alonso Garrido Sara H Goldemberg Israel D Algranati |
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Institution: | 1. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires, Argentina;2. Instituto de Investigaciones Bioquímicas ‘Fundacion Campomar’, and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Antonio Machado 151, Buenos Aires Argentina |
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Abstract: | In order to study the intracellular polyamine distribution in Escherichia coli, 13C-NMR spectra of 1,4-13C]putrescine were obtained after addition of the latter to intact bacteria. The 13C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using 13C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound 13C]putrescine could be displaced by addition of an excess of 12C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with 1,4-13C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements η, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T1 values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, τc = 4·10?7 s for the latter. T1 and τc value for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules. |
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Keywords: | Putrescine distribution (E coli) |
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