RESUMO
One isoform of potato (Solanum tuberosum L., cv. Spunta), type L phosphorylase (EC 2.4.1.1), exhibiting primer independent activity appears to be tuber-specific. However, this activity can also be modulated by exogenous sucrose in storage as well as in non-storage organs. Primer independent phosphorylase (PIPh) activity in microtubers and shoots of in vitro plantlets was found to be much higher than in tubers and shoots of soil-grown plants. Detached leaves of soil-grown plants showed an increase in PIPh activity as well when incubated in sucrose-containing Murashige-Skoog (MS) medium. This increase was always accompanied by a rise in starch content. The presence of metabolizable carbohydrates in the growth or incubation medium are likely to be responsible for the observed rise in PIPh activity. In vitro microtubers and micropropagated plantlet organs (shoots and roots) exhibited a correlation between measurable PIPh activity and presence of enzyme protein, as judged by Western blot analysis using anti-potato tuber type L phosphorylase antibody. Therefore, in addition, to be developmentally regulated (tuber-specific accumulation), PIPh activity associated with the tuber type L isoform might be under a form of metabolic regulation.
Assuntos
Isoenzimas/metabolismo , Fosforilases/metabolismo , Solanum tuberosum/enzimologia , Isoenzimas/análise , Fosforilases/análise , Solanum tuberosum/crescimento & desenvolvimento , Amido/metabolismo , Sacarose/metabolismo , Distribuição TecidualRESUMO
A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.