The complex behavior of the mixture galactosidase/protease in heterogeneous systems

SANCHEZ, JM; PERILLO, MA

UBA-Bs, As. Argentina

Congreso; Satellite of XIV International Biophysics Congress; 2002

SAB

Previously we demonstrated that, in the presence of model membranes, the activity of b-galactosidase against a soluble substrate could be differentially modulated through enzyme-membrane interactions, according to the enzyme possibility to penetrate or just be adsorbed to a dimensionality restricted space. Surface topology was crucial in determining the direction of the modulating effects and the differences in surface curvature, and thus in surface molecular packing and hydration,  accounted for the  effects observed as the main modulating factor. (Coll.&Surf. 24,21,2002). In addition, due to enzyme-membrane interactions, b-Galactosidase activity was preserved in aggressive media like at extreme pHs and temperatures or in the presence of proteolytic enzymes (Sanchez and Perillo, WSBiomembranes,2001). In the present work we investigated the concomitant modulation of protease and b-galactosidase activities by multilamellar vesicles (MLVs) of soybean phosphaditylcholine (PC). The reactions studied were: a) the hydrolysis of ortho-nitrophenol-b-D-galactopiranoside (ONPG) to ortho-nitrophenol (ONP) catalyzed by b-galactosidase, by the method of Wallenfels and Malhota, Adv.Carbohyd.Chem. 16,239,1975) and b) the hydrolysis of bovine serum albumin (BSA) to free aminoacids, catalyzed by protease type IV from Sigma Chem.Co., by the method of Mirsky et al. (in: Henry et al., 1978). In some experiments, reaction a was studied in the presence of protease; b-galactosidase was either encapsulated or not encapsulated in the MLVs. Different combinations of the following mass ratios: PC/b-galactosidase, PC/protease and  protease/b-galactosidase (in the first reaction) and PC/BSA, PC/protease and protease/BSA (in the second reaction), were tested. Particular samples of b-galactosidase containing or not either PC and/or protease were also submitted to PAGE-SDS. Our results indicated that: i) the activity of b-galactosidase was truly protected against proteolysis only at high PC/b-galactosidase  ratio (@107) and low PC/protease ratio (@ 6.5); in this condition, the surface-mediated b-galactosidase activation was also expressed. Protease could be either activated by the immobilization of the substrate (at relatively low PC/protease ratio (@ 78) and high PC/BSA (@ 117-1164) or inhibited by the enzyme adsorption to the lipid-water interface (at PC/protease ratio@790) (this inhibition disappeared in the presence of an excess of Triton X-100). As a consequence a complex  pattern of b-galactosidase activity was obtained in the PC/b-galactosidase-PC/protease phase space.