Characterization of molecular aggregates of alpha 1 beta 1-integrin and other rat liver membrane proteins by combination of size-exclusion chromatography and chemical cross-linking.
Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregates 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 aggregates was investigated by two approaches. First, after modest detergent extraction, 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 could be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied detergents partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggregates. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpropionate) was combined with the performance of denaturating SEC. Under such optimized some high-molecular-mass complexes of all model proteins consisting of unknown components could also be detected. Taken together, non-denaturating SEC and chemical cross-linking in combination with denaturating SEC represent methodological approaches for the characterization of protein aggregates.
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000818:Animals; D015703:Antigens, CD; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D003432:Cross-Linking Reagents; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D039222:Integrin alpha1beta1; D016023:Integrins / Q000737:chemistry*; D008099:Liver / Q000737:chemistry* / Q000201:enzymology; D008565:Membrane Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D051381:Rats
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D039222:Integrin alpha1beta1; D016023:Integrins; D008565:Membrane Proteins; D018819:Dipeptidyl Peptidase 4; D000251:Adenosine Triphosphatases