BETA-GALACTOSIDASE ACTIVITY MODULATION BY ADSORPTION OR PENETRATION IN DIMENSIONALITY RESTRICTED SPACES

JULIETA M.SANCHEZ AND MARIA A.PERILLO

Campiñas. San Pablo Brasil

Congreso; IV congreso de Biofísica del Conosur; 2000

SBBF

We investigated if in complex environments, like those where beta-galactosidase activity is usually assayed, the kinetics of soluble substrates hydrolysis could be modulated through enzyme-surface interactions. Kinetic parameters were determined using ortho-nitrophenyl-beta-D-galactopiranoside (ONPG) as substrate, in the absence or presence of pure phosphatidyl cholines (PCs) or PCs:cholesterol bilayers and detergents (monomers or micelles), alone or in mixtures with PC. At low [PC] KM and Vmax decreased. At high [PC], condensed packing increased both kinetic parameters and low packing increased only Vmax. Kinetic parameters were unaffected by ionic detergents as monomers but, in micelles they increased KM and decreased Vmax. Above their critical micellar concentration, non-ionic detergents reverted PC’s effects on Vmax and the ionic ones expressed their own inhibitory effects. Non-enzymatic interfacial hydrolysis of ONPG was discarded. Enzyme-membrane interaction was confirmed using monomolecular-layers at the air-water- interface. Concluding, beta-galactosidase activity might be modulated according to the enzyme possibility to penetrate or just be adsorbed to a dimensionality restricted space. Partitioning of reaction substrate and product towards the membrane may also play a role in the dynamics of the overall process. Supported by FONCYT, CONICOR and SeCyt-UNC.