Photoaffinity labeling of lactose synthase with a UDP-galactose analogue.
A photoaffinity analogue of UDP-galactose, 4-azido-2-nitrophenyluridylyl pyrophosphate (ANUP), has been synthesized for the investigation of the binding topography of alpha-lactalbumin on galactosyltransferase. Results obtained from steady state kinetics show that ANUP is an effective competitive inhibitor against UDP-galactose in the reactions of lactose and N-acetyllactosamine syntheses. The specific binding of ANUP to the UDP-galactose-binding site is further demonstrated by its ability to facilitate the formation of the lactose synthase complex on solid supports, either alone or in the presence of glucose or N-acetyl-glucosamine. ANUP inactivates galactosyltransferase on irradiation. One mole of ANUP was incorporated per mol of enzyme inactivated. This process is Mn2+-dependent and can be prevented by UDP-galactose. Glucose and N-acetylglucosamine render only partial protection. Photoaffinity labeling of lactose synthase either free in solution or immobilized on Sepharose does not result in any reduction of the alpha-lactalbumin modifier activity. In addition, no incorporation of radioactivity into alpha-lactalbumin was observed when radioactive ANUP was used, whereas galactosyltransferase was labeled. These data indicate that alpha-lactalbumin does not bind to galactosyltransferase in the region of the ANUP site, suggesting that the location of protein-protein interaction between the two subunits of lactose synthase may be removed from the UDP-galactose-binding domain.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology*; D002417:Cattle; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism; D007700:Kinetics; D007788:Lactose Synthase / Q000037:antagonists & inhibitors*; D008024:Ligands; D008892:Milk / Q000201:enzymology; D010782:Photolysis; D014531:Uridine Diphosphate Galactose / Q000031:analogs & derivatives*; D014539:Uridine Diphosphate Sugars
D000345:Affinity Labels; D001386:Azides; D004800:Enzymes, Immobilized; D008024:Ligands; D014539:Uridine Diphosphate Sugars; D014531:Uridine Diphosphate Galactose; C039746:4-azido-2-nitrophenyluridylyl pyrophosphate; D005700:Galactosyltransferases; D007788:Lactose Synthase