Use of concanavalin A as a topographical probe for protein-protein interaction. Application to lactose synthase.
Galactosyltransferase (EC 2.4.1.38) has been shown to bind to Con A-Sepharose. Concentrations of methyl-alpha-mannoside greater than 0.7 M were required to release the enzyme from the immobilized lectin. Molecular weight determination by gel filtration revealed that galactosyltransferase formed a 1:1 complex with concanavalin A. Preincubation of the enzyme with excess concanavalin A did not affect its catalytic activity either in the presence or absence of alpha-lactalbumin. The galactosyltransferase-concanavalin A complex was retained on an alpha-lactalbumin-Sepharose column in the presence of N-acetylglucosamine and manganese chloride and was eluted from the column in their absence. Galactosyltransferase immobilized onto a Con A-Sepharose was still active either in the presence or absence of alpha-lactalbumin. Lactose synthase activity was also observed when the galactosyltransferase-concanavalin A complex was assayed with alpha-lactalbumin immobilized on Sepharose. These data indicate that the carbohydrate moiety of galactosyltransferase is involved in neither the catalytic process nor the binding of alpha-lactalbumin and must be linked to the enzyme at a location where it does not present any steric hindrance on the binding of concanavalin A, either free or immobilized on Sepharose.
D000818:Animals; D002417:Cattle; D003208:Concanavalin A; D004789:Enzyme Activation / Q000187:drug effects; D004800:Enzymes, Immobilized; D005700:Galactosyltransferases; D007788:Lactose Synthase / Q000302:isolation & purification*; D008892:Milk / Q000201:enzymology; D011485:Protein Binding
D004800:Enzymes, Immobilized; D003208:Concanavalin A; D005700:Galactosyltransferases; D007788:Lactose Synthase