CHARACTERIZATION OF MOLECULAR AGGREGATES OF ALPHA(1)BETA(1)-INTEGRIN AND OTHER RAT-LIVER MEMBRANE-PROTEINS BY COMBINATION OF SIZE-EXCLUSIONCHROMATOGRAPHY AND CHEMICAL CROSS-LINKING

Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregat es remains difficult; especially intermolecular complexes of membrane proteins tend to dissociate or artificially aggregate during detergent extraction out of membranes, Thus, the existence of protein aggregate s was investigated by two approaches. First, after modest detergent ex traction, the presence of three well characterized rat liver membrane proteins, alpha(1) beta(1)-integrin, dipeptidyl aminopeptidase IV (DPP IV) and cell-CAM 105 (CAM = cell adhesion molecule), in aggregates co uld be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied deterge nts partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggrega tes. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpr opionate) was combined with the performance of denaturating SEC. Under such optimized conditions the expression of alpha(1) beta(1)-integrin as heterodimer and DPP IV as homodimer was confirmed. In addition, so me high-molecular-mass complexes of all model proteins consisting of u nknown components could also be detected. Taken together, non-denatura ting SEC and chemical cross-linking in combination with denaturating S EC represent methodological approaches for the characterization of pro tein aggregates.