19] Modulation of membrane protein function by surface potential

Abstract

This chapter correlates quantitatively a certain surface potential to its effect on definite kinetic parameters of membrane-bound enzymes or carriers. Thus, apart from the preparation of suitable reconstituted proteoliposomes forming the basis for the experiments, both the appropriate determination of surface potential or surface charge and the analysis of the characteristic kinetic constants of the enzymatic or transport process are described. For experiment it is necessary to use a definite lipid composition. Liposomes can be prepared that necessitate to incorporate functionally active proteins into membranes. Certain detergent removal techniques are used, such as dialysis, column procedures, and removal by hydrophobic absorption. For measurement of surface potential two methods are used. The use of 2-p-toluidinylnaphthalene 6-sulfate as charged fluorescent distribution probe is convenient for determining the surface potential of proteoliposomes. The second method used for the determination of surface potential is based on the pK shift of the lipophilic pH indicator alkylumbelliferone. To make a quantitative correlation of various kinetic parameters with the observed surface potential, one must always be aware of the complexity of possible interactions and influences at certain steps within a complete reaction cycle of an enzyme or carrier system.