IIBYT   23944
INSTITUTO DE INVESTIGACIONES BIOLOGICAS Y TECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
In vitro and in vivo -Gal self-assembling improve the enzymatic activity and stability
Autor/es:
MARÍA A. PERILLO; SANDRA S. FLORES; MARIA V. NOLAN; PEDRO D CLOP; JULIETA M. SANCHEZ
Lugar:
San Luis
Reunión:
Encuentro; XLVIII Reunion Anual de la Sociedad Argentina de Biofísica.; 2019
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
In our laboratory we are interested in describing the functional plasticity of proteins in their self assembled states driven by oligomerization and/or aggregation.The objective of this work is to study how the oligomeric/aggregated states of E. coli beta galactosidase (β-Gal) modulate the enzymatic activity with respect to the monomeric state.We achieved self-assembled β-Gal by two strategies: In vitro; controlling the presence of MgCl2 in the β-Gal solution, and in vivo: isolating aggregate protein in the form of inclusion bodies (IBs) from E. coli. The presence of oligomers and/or aggregates were evidenced by means of analytical ultracentrifugation and by quasi-elastic light scattering.In all the cases the activity was evaluated using lactose as a substrate and quantifying glucose as the hydrolysis product. The reaction conditions were 37° C and pH 6.8 maintained by 0.1 M phosphate buffer. In all self-assembled states, we measured the kinetic parameters by non-linear regression of the experimental data using the minimum square method. Besides we also evaluated the optimum temperature and pH of the enzyme in each format.We prove that Mg+2 is able to stabilize the tetrameric forms of the enzyme but not higher oligomeric states. We could produce and isolate functional IBs of β-Gal of around 1000 nm. We also demonstrate that the catalytic activity increases when the enzyme is self-assembled. On the other hand, studies of β-Gal activity against pH and temperature are similar in the absence or in the presence of MgCl2. However, when we study the pH and temperature profiles of the β-Gal in IBs, we observe their higher stability compared to the soluble enzyme.Our results contribute to highlighting the effect of protein-protein interaction on the protein functionality beyond the origin and dimension of the protein supramolecular structure.This work was partially supported by grants from SeCyT-UNC, ANPCyT and CONICET