Invited Discussions Microenvironmental Effects on Enzyme Activity

One of the problems to be considered in evaluating the results of experiments involving fragmentation of cells and subcellular organelles and fractionation of the particles and supernatants obtained, is that enzymic activities measured by a standard assay at various stages of the procedure may not provide a true measure of the amount of a given enzyme which is present. In some cases the reason for this may be that the enzyme is localized inside a com-partment, for example within a vesicular organelle, which the substrate cannot penetrate. However, more subtle microenvironmental effects may also play a role. An enzyme which is bound to or embedded in a lipoprotein membrane is situated in a milieu different from that of the same enzyme in aqueous solution. The bound enzyme may, therefore, display an activity different from that of the soluble enzyme, as a result of local charge effects or of the lipid character of the membrane. Moreover, diffusion of substrates and products to and from a membrane-bound enzyme may be affected owing to the presence of unstirred layers around the membrane fragment. At the Weizmann Institute we have been engaged in studying enzymes bound to insoluble polymeric arriers, and to synthetic membranes, as simple model systems for native particulate nzymes (see, for example, reference 1). In the following, I want to describe briefly one type of anomalous enzymic activity observed in a synthetic membrane, and to show how the explanation for the behavior of this system could, in its turn, be used to explain the be-havior of a natural membrane-bound enzyme. The synthetic enzyme membrane system under consideration was pre-pared by impregnating a highly porous collodion membrane with a solution of crystalline papain, and then chemically cross-linking the absorbed en-zyme with the bifunctional reagent bisdiazobenzidine disulfonic acid (2, 3). The membranes used in the experiments I will describe were about 400 thick and contained layers of enzyme 70 /, thick on each side, colored by reaction with the cross-linking agent (Fig. 1). The papain-collodion mem-brane contained about 1 mg cf enzyme per cm 2. These membranes were very 50