Eric Selegny

Monoenzymatic model membranes : Diffusion-reaction kinetics and phenomena

Summary New procedures of enzyme binding to membrane matrices are described. These systems are used to build models of metabolite transfer in and across biological membranes and particles. Two methods have been used successfully. The first one consists of bonding proteins inside a matrix ; the second one is a coreticulation of enzymes with an inactive protein. Both methods maintain an important fraction of the initial enzymatic activity. The yields observed reach 80 p. cent. Using these procedures, various enzymes have been efficiently bound. Thus, their resistance to denaturation and proteolysis is increased. Several properties remain unchanged : specificity, activation energy of the reaction, and enzyme-substrate affinity. A greater pH-dependence of enzyme activity after fixation is observed. The function of the enzymatic membranes obtained is controlled simultaneously by two phenomena : enzymatic reaction and diffusion of metabolites. The respective and mutual effects of these two phenomena are analyzed. Our mathematical analysis of these systems gives rise to equations which coincide with and explain our experimental results.

Structured bienzymatical models formed by sequential enzymes bound into artificial supports: Active glucose transport effect

SummaryTwo different artificial membrane systems bearing two built-in sequential enzymes are studied and compared in this communication.The first is a nonstructured membrane bearing two mixed enzymes: β-galactosidase and glucose-oxidase. Its use enables a mathematical model to be formulated describing the heterogeneous phase kinetics of a bienzymatic system. The second is a multi-layer membrane system in which the structural dissymmetry involves a spatial orientation of the reacting metabolites, resulting in active glucose transport.The latter system consists of two active leaflets, the first phosphorylating glucose (hexokinase+ATP), the second dephosphorylating glucose-6 phosphate (phosphatase). On either side of this system, a perm-selective proteic layer allows the passage of glucose but not of glucose-6 phosphate. When positioned between two compartments containing glucose, such a membrane accumulates glucose on its phosphatase side, while degrading ATP.The accumulation of glucose as a function of the initial concentration shows the classical saturation of the transport system. Fructose competes with glucose transport.The chemical balance of these two reactions has the appearance of hydrolysis of ATP. Vectorial catalysis is a result of the dissymmetry in distribution of active sites and can be explained by an oscillatory concentration profile of glucose inside the membrane.The bienzymatic mechanism, a model of which is given here, is valid for any thickness of active layers and applicable to a system where both active sides are part of the same molecule as soon as it forms a uniformly oriented monolayer throughout the membrane structure.