On the interaction of alpha-lactalbumin and galactosyltransferase during lactose synthesis.
The regulatory effect of alpha-lactalbumin in the lactose synthase system has been ascribed to its reversible association with a complex of galactosyltransferase with Mn2+ and UDP-galactose, prior to the binding of monosaccharides; the resulting complex has a higher affinity for various monosaccharides. Two steps in the postulated catalytic cycle have been investigated; UDP-galactose binding to enzyme-Mn2+ by equilibrium dialysis and alpha-lactalbumin binding to enzyme-Mn2+-UDP-galactose by sedimentation velocity and kinetics. There is a single binding site for UDP-galactose on the enzyme-Mn2+ complex, and the dissociation constant for UDP-galactose from enzyme-Mn2+-UDP-galactose was found to be 72 muM at 37 degrees. The formation of a complex between galactosyltransferase and alpha-lactalbumin in the presence of Mn2+ and UDP-galactose was observed as an increase in sedimentation coefficient of enzyme activity So20,w from 3.25 +/- 0.03 in the absence of alpha-lactalbumin to 4.22 +/- 0.03 at saturating concentrations of alpha-lactalbumin, a value closely similar to that of a cross-linked 1:1 complex of the proteins under the same conditions (4.35 +/- 0.03). No interaction was observed in the absence of substrates or with UDP-galactose and EDTA. From the ultracentrifuge data and steady state kinetics, dissociation constants for alpha-lactalbumin from the enzyme-Mn2+-UDP-galactose-alpha-lactalbumin complex were determined at several temperatures and salt concentrations. These showed good internal agreement. The free energy change delta G degrees for the association of the two proteins is calculated, and the results are discussed in relation to the nature of the interaction.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003126:Colostrum / Q000201:enzymology; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D007785:Lactose / Q000096:biosynthesis*; D007788:Lactose Synthase / Q000378:metabolism; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008892:Milk / Q000201:enzymology; D008970:Molecular Weight; D011485:Protein Binding; D014531:Uridine Diphosphate Galactose
D046911:Macromolecular Substances; D014531:Uridine Diphosphate Galactose; D008345:Manganese; D007768:Lactalbumin; D005700:Galactosyltransferases; D007788:Lactose Synthase; D007785:Lactose