The Interaction of α-Lactalbumin and the A Protein of Lactose Synthetase

Lactose synthetase is made up of a galactosyl transferase (the A protein) and α-lactalbumin which controls the specificity of the reaction by regulating the affinity of the system for acceptor sugars. We have purified the A protein from bovine skim milk so that it is free of contaminating proteins. This preparation is a single chain glycoprotein of molecular weight 44,000 with a sedimentation coefficient of 3.2 S. The enzyme behaves on disc gel electrophoresis as a family of closely related components all of which are enzymically active and contain carbohydrate. A rapid and sensitive method of studying macromolecular interactions is described. In this procedure the band forming cell of Vinograd is used and the migration of a band of A protein through solutions of α-lactalbumin is followed in the analytical ultracentrifuge equipped with a photoelectric scanner. These experiments show that a complex is formed between A protein and α-lactalbumin which contains one molecule of each protein component. The complex is stable enough to be observed only in the presence of one of the substrates of the enzyme such as UDP-galactose or N-acetylglucosamine. Binding of tetranitromethane-treated α-lactalbumin to A protein in the presence of UDP-galactose could be demonstrated by conventional sedimentation velocity techniques using absorption optics at 430 nm.