A cross-linked complex between horse pancreatic lipase and colipase.
The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.
Chaillan
C
C
CNRS, Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
Rogalska
E
E
Chapus
C
C
Lombardo
D
D
D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides; D003089:Colipases; D003432:Cross-Linking Reagents; D006736:Horses; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007477:Ions; D008049:Lipase; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D011506:Proteins; D013329:Structure-Activity Relationship
D002234:Carbodiimides; D003089:Colipases; D003432:Cross-Linking Reagents; D007477:Ions; D011506:Proteins; D008049:Lipase