title: A comparison of the interactions of galactosyltransferase with a glycoprotein substrate (Ovalbumin) and with alpha-lactalbumin.
abstract: Sedimentation velocity ultracentrifugation and other procedures have been used to investigate macromolecular interactions of bovine colostrum galactosyltransferase with a glycoprotein substrate (ovalbumin) and with the lactose synthase regulatory protein, alpha-lactalbumin. The determination of equilibrium binding constants for these interactions and the effects of ligands and combinations of ligands on the equilibria have clarified several aspects of the mechanism of galactosyltransferase and its regulation by alpha-lactalbumin. 1. The attachment of Mn2+ at the tight binding site on galactosyltransferase (site I, Kd 2.3 muM) is an essential prerequisite for interactions with ovalbumin and with alpha-lactalbumin. 2. The attachment of Mn2+ or Ca2+ at the weaker metal binding site (site II, Kd 1 to 2 mM) does not significantly affect the interaction of galactosyltransferase with either protein. This is consistent with the hypothesis derived from kinetic studies that site II is functionally connected with the binding of UDP-derivatives. 3. While the binding of ovalbumin to galactosyltransferase in the presence of Mn2+ alone can be observed by ultracentrifugation, this interaction is too weak to cause binding of galactosyltransferase to ovalbumin-Sepharose. Binding to ovalbumin-Sepharose could only be detected by affinity chromatography in the presence of both Mn2+ (10 mM) and UDP (0.3 MM). Sedimentation studies showed that the association of galactosyltransferase with ovalbumin is pressure-dependent, and that the presence of UDP in the complex increases the equilibrium association constant by a factor of 46. The enzyme Mn2+-UDP-ovalbumin complex has unusual hydrodynamic properties. 4. The presence of saturating concentrations of UDP-galactose potentiates the binding of alpha-lactalbumin at high concentrations of Mn2+, as shown by a 25-fold increase in the association constant. Competitive inhibition by alpha-lactalbumin, with respect to ovalbumin that is observed by steady state kinetics, is attributed to the mutally exclusive binding of the proteins with an enzyme complex containing Mn2+ and UDP-galactose. 5. Monsaccharides (N-acetylglucosamine and glucose) strongly enhance the binding of alpha-lactalbumin to enzyme complexes containing Mn2+, in the presence or absence of UDP-glucose. The binding of alpha-lactalbumin and monosaccharide to form  enzyme complexes containing both is random, and evaluation of the four associated equilibrium constants shows that the binding is strongly synergistic. 6. Although the significance of some of the many equilibria studied cannot be ascertained, it appears than an element of randomness may be present in reactions catalyzed by galactosyltransferase...
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D007768:Lactalbumin; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D010047:Ovalbumin; D011247:Pregnancy; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D008345:Manganese; D010047:Ovalbumin; D007768:Lactalbumin; D005700:Galactosyltransferases; D002118:Calcium