Specific precipitation of enzyme by its substrate: the α-amylase-macrodextrin complex

Abstract

Abstract Addition of glycogen to a cold aqueous pancreatin extract caused the specific precipitation of α-amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1). The precipitate contained glycogen dextrins bound to the enzyme. The nature of this interaction was further studied with crystalline pancreatic α-amylase and fractionated α-limit dextrins. The enzyme formed an insoluble complex only with dextrins which had a polymeriazation degree higher than 22. For maximal precipitation dextrins with a polymerization degree higher than 62 were required. Under optimal conditions 80% of the dextrin or the enzyme could be incorporated into the insoluble complex. Precipitation was inhibited by excess dextrin as well as by excess enzyme. The mechanism of precipitation appears to be identical to that operating in the antigen-antibody precipitin reaction. It is thus indicated that α-amylase and the dextrin, each possessing more than one combining site can form a lattice structure in which many molecules of both reactants are linked to each other. “Calcium-free” α-amylase, which is known to be enzymically inactive, failed to yield a precipitate. The capacity to form the insoluble complex with dextrin could be restored by adding either Ca, Sr or Ba. The significance of these findings in the study of enzyme-substrate interactions is discussed.