Use of concanavalin a as a topographical probe for protein-protein interaction Application to lactose synthase

Abstract

Galactosyltransferase (EC 2.4.1.38) has been shown to bind to Con A-Sepharose. Concentration of methyl-α-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 α-lactalbumin. The galactosyltransferase-concanavalin A complex was retained on an α-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 α-lactalbumin. Lactose synthase activity was also observed when the galactosyltransferase-concanavalin A complex was assayed with α-lactalbumin immobilized on Sepharose. These data indicate that the carbohydrate moiety of galactosyltransferase is involved in neither the catalytic process nor the binding of a α-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.