RESUMO
In order to comprehend the function of a particular protein, identification of the interacting protein partners is a useful approach. Co-immunoprecipitation (Co-IP) is employed to test physical interactions between proteins. Specific antibodies or antibodies against tagged versions can be used to immunoprecipitate the proteins. In this chapter, we describe a method to carry out Co-IP using recombinant membrane proteins expressed in yeast microsomal fractions.
Assuntos
Anticorpos/química , Imunoprecipitação/métodos , Mapeamento de Interação de Proteínas/métodos , Proteína Quinase C/isolamento & purificação , Solanum lycopersicum/genética , Western Blotting , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ligantes , Solanum lycopersicum/enzimologia , Microssomos/química , Ligação Proteica , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.
Assuntos
Proteína Quinase C/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Western Blotting , Bovinos , Cromatografia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Durapatita , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ésteres de Forbol/metabolismo , Fosforilação , Proteína Quinase C/imunologia , Proteína Quinase C/isolamento & purificação , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.