DEMONSTRATION OF ENZYME ASSOCIATIONS BY COUNTERMIGRATION ELECTROPHORESIS IN AGAROSE-GEL

We propose a method to study multienzyme complex formation in vitro ba sed on nondenaturing agarose gel electrophoresis. The enzymes with dif ferent isoelectric points (pI) were loaded at the opposite ends of the same lane of agarose gel and electrophoresis was performed at a pH va lue intermediate between their pI's. In cases where a complex of the e nzymes was formed, an additional protein band of low electrophoretic m obility was found corresponding to the point where they crossed on the gel. This band contained both enzyme activities. The method was used to demonstrate association between two enzymes of the mitochondrial ci tric acid cycle, malate dehydrogenase and citrate synthase, and betwee n the lysosomal hydrolases, beta-galactosidase and cathepsin A. Relati ve proportions of free and bound enzymes after electrophoresis suggest that interaction between the mitochondrial enzymes is relatively weak compared to that of lysosomal hydrolases. Microdensitometric scanning of countermigration electrophoresis gels was used to determine the st oichiometry of components in the complex. (C) 1994 Academic Press, Inc .