Heterogeneous chromatographic behaviour or soluble RNA replicase from healthy and virus-infected tobacco leaves: an improvement of the purification methodology.
During purification of soluble RNA replicase from healthy and alfalfa mosaic virus-infected tobacco leaves, crude extracts were chromatographed on DEAE-Sephadex A-25, and three RNA-dependent RNA polymerase activities were obtained; one of these was previously characterized as a true RNA replicase, the remaining two were not studied (Chifflot et al., 1980, Virology 100, 91). We now demonstrate that all of these activities correspond to the same RNA replicase, complexed or not to cellular or viral RNA. Upon chromatography on DEAE-Sephadex A-25, the free replicase did not bind to the gel at low ionic strength, whereas the RNA-replicase complexes were bound to the gel through the medium of RNA. Increasing the ionic strength allowed the dissociation of the complexes and elution of the replicase only. From this observation, a new and rapid purification procedure combining all these activities and yielding large amounts of replicase was developed; the first main step of purification was chromatography on Blue-Sepharose CL6B under conditions conductive to the dissociation of RNA-replicase complexes, and thus maximal adsorption of the replicase. The enzyme was then eluted by increasing the ionic strength and was further purified on coupled DEAE-Sephadex A-25 and phosphocellulose columns. The DEA-Sephadex A-25 was used to bind the remaining RNA, while the replicase passed through, and bound to the cationic ion exchanger. The final replicase preparations which were obtained were very stable and had a purification factor of 1100-1400. The recovery averaged 70% and specific activities were much higher than those already described for similar enzymes from healthy or virus-infected tobacco leaves.
D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D000455:Medicago sativa; D009029:Mosaic Viruses / Q000254:growth & development*; D010947:Plants, Toxic; D012313:RNA; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000382:microbiology
D012367:RNA, Viral; D012313:RNA; D012316:RNA Nucleotidyltransferases; D012324:RNA-Dependent RNA Polymerase