INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Biophysical characterization of a perivitellin in the freshwater snail Pomacea canaliculata
Autor/es:
FRASSA,V.; DREON MS,; CEOLIN,M.; HERAS, H
Lugar:
Rosario
Reunión:
Congreso; XXXV Reunión Anual Sociedad Argentina de Biofísica (SAB); 2006
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
Pv2, together with ovorubin, are the major high MW lipo-glycoproteins present in the eggs of P. canaliculata. This autochthonous freshwater snail has remarkable economical and health aspects, especially in Far East Asia, as a rapidly expanding rice field pest. We have previously characterized Pv2 regarding its lipid, glucid and protein moieties, described the cellular type involved in its synthesis, tissular distribution, and identified some of Pv2 functions such as its contribution to energy metabolism, structural precursors and defenses for the developing embryo. This protein is a very high-density lipoprotein (VHDL) of 400 KDa, 3.75% lipids and 2.5 % carbohydrates composed of two subunits of 67 and 31 KDa [1, 2, 3]. Different structural parameters of Pv2 were studied by Small Angle X-ray Scattering (SAXS) to evaluate the stability and unfolding mechanism, using urea (0 to 6M) as chaotropic agent and temperature (20 to 80°C). To gain structural insight on the disassembly/denaturing process we analyzed the evolution of the gyration radius obtained from Guinier plots and the shape and position of the Kratky-plots. In order to assay the relevance of the disulfide network on the stability of the oligomer, we also performed non-denaturing PAGE and UV-Vis spectroscopy on samples treated with the Ellman´s reagent. From the SAXS results we were able to obtain a low resolution 3D model with a gyration radius of 44 ± 1 Å for the native protein, being the first time a structural model for this protein was built. A small but significant decrease of the gyration radius was observed for urea concentrations up to 1.5M indicating the disassembling of the oligomer. At concentrations higher than 2M urea, the gyration radius of the protein began to grow up, reaching 66 ± 5 Å at 6M urea, a value nevertheless well below the expected one for the fully denatured subunits. Pv2 also underwent steep conformational changes between 50 and 60°C, as indicated by the Kratky plots. Compared with ovorubin, Pv2 showed a higher sensitivity to thermal and urea denaturalization. The complementary roles the 2 perivitellins play during embryogenesis such as the presence of energy-providing triacylglycerols in Pv2 and an antioxidant and antiprotease activities in ovorubin, seems to be correlated with their contrasting structural stability.