RESUMO
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.
Assuntos
Oligopeptídeos/química , Conformação Proteica , Dobramento de Proteína , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/análise , Proteínas Fúngicas/química , Hexoquinase/análise , Hexoquinase/química , Substâncias Macromoleculares , Oligopeptídeos/análise , Fosforilase a/análise , Fosforilase a/química , Fosforilases/análise , Fosforilases/química , Pressão , Desnaturação Proteica , Saccharomyces cerevisiae/química , UreiaRESUMO
A protein kinase C activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns giving two peaks of kinase activity which were eluted at 0.1 and 0.15 M NaCl. The first activity peak requires Ca2+ and phosphatidylserine for activity. Further kinase purification was performed by chromatography on phenyl Sepharose columns. In these columns the enzyme activity was adsorbed in the presence of Ca2+ and eluted with a EGTA-containing buffer. T. cruzi protein kinase C activity preferentially phosphorylated histone H1. It was stimulated by diacylglycerol and phorbol myristate acetate, and inhibited by polymyxin B and staurosporine. After subcellular fractionation and epimastigote cells, the kinase was found to be associated with microsomal and cytosolic fractions.
Assuntos
Proteína Quinase C/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Cálcio/farmacologia , Cromatografia em Agarose , Cromatografia DEAE-Celulose , Citosol/enzimologia , Diglicerídeos/farmacologia , Ácido Egtázico/farmacologia , Histonas/metabolismo , Microssomos/enzimologia , Fosfatidilserinas/farmacologia , Fosforilação , Proteína Quinase C/isolamento & purificação , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
A method is presented for wavelength calibration of spectrofluorometer monochromators. It is based on the distortion that the characteristic absorption bands of glass filters (holmium or didymium oxide), commonly used for calibration of spectrophotometers, introduce in the emitted fluorescence of fluorophores like indole, diphenyl hexatriene, xylene or rhodamine 6G. Those filters or a well characterized absorber with sharp bands like benzene vapor can be used for the same purpose. The wavelength calibration accuracy obtained with this method is better than 0.1 nm, and requires no modification in the geometry of the spectrofluorometer sample compartment.