Lactose synthetase. The isolation and characterisation of the protein-protein complex.

Abstract

Galactosyl transferase and alpha-lactalbumin were concurrently isolated from bovine milk. The galactosyl transferase had an S20,w of 3.0 S and D20,w of 60 mum2/s. It exists as a monomer of 46000 molecular weight, as determined by sedimentation equilibrium and dodecylsulphate-polyacrylamide electrophoresis. Aggregation of the enzyme was promoted by N-acetylglucosamine. Sedimentation velocity experiments show an association between galactosyl transferase and alpha-lactalbumin in the presence of N-acetylglucosamine. In contrast, UDP-galactose, UDP or lactose do not promote protein-protein association. A complex between galactosyl transferase and alpha-lactalbumin was isolated by gel filtration in the presence of excess alpha-lactalbumin and either N-acetylglucosamine or glucose. The complex was stable over a range of concentrations of these components. The complex is a discrete homogeneous entity with a molecular weight of 60000, corresponding to one molecule of galactosyl transferase and one molecule of alpha-lactalbumin. The estimated association constants for the ternary complexes of the two proteins and either of the sugars, suggest that alpha-lactalbumin enhances equally the binding of N-acetylglucosamine or glucose to the galactosyl transferase.