Application of short column gel permeation in the study of protein-protein interactions

A simple and rapid procedure based on the gel filtration principle is described together with its applicability to the study of protein-protein interactions including subunit-subunit and enzyme-enzyme interactions. Using this procedure, it is shown that phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) interact with a stoichiometry of one PGK molecule combining with one monomeric subunit of GPDH. This interaction has been observed with both enzymes being from the same, as well as from different, species. The Kd values for rabbit muscle PGK and porcine muscle GPDH complex and that for the rabbit muscle PGK and yeast GPDH complex are found to be (4.5 +/- 2.0) x 10(-7) M and (6.5 +/- 1.7) x 10(-7) M, respectively. The specificity of bienzyme association is stronger when enzymes are from the same species than when they are from different species.