RESUMEN
Analysis of enzymatic activity in polyacrylamide gel is based on highly effective separation of proteins by SDS-electrophoresis with their subsequent renaturation and detection of enzymatic activity. This method was used to study an expression of DNAases in culturing of cells HEK293, NIH 3T3, U937. We have found that in HEK293 cells the nucleases with molecular weights 47 and 45 kDa were expressed. The localization of DNAases in the cell nuclei was shown as well. Induction of apoptosis in HEK293 cells increase the level of p47 DNAase and causes the expression of novel 50 kDa DNAase. We suggested that those discovered DNAases could take part in apoptotic DNA degradation.
Asunto(s)
Desoxirribonucleasas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Renaturación de Proteína , Células 3T3 , Animales , Línea Celular , Núcleo Celular/enzimología , Humanos , Ratones , Peso Molecular , Células U937RESUMEN
The changes in protein-protein interactions mediated by SH3 domain of the regulatory p85 alpha subunit of phosphatidylinositol 3-kinase (Pl 3-kinase) in the course of herbimycin A-induced erythroid differentiation of the human erythroleukemia cell line K562 have been analyzed. Binding assay was performed in vitro using a recombinant form of SH3 domain of p85 alpha, conjugated with glutathione-S-transferase. pTyr-containing 210, 116, 52 and 46 kDa proteins, binding of which are modulated in differentiated cells, were identified and binding dynamics was analysed. The obtained data on binding the specific pTyr-containing proteins with regulatory subunit of Pl 3-kinase testify about the coordinated control of Pl 3-kinase signalling pathway in the course of herbimycin A-induced K562 cells differentiation.