Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative.
The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D003126:Colostrum / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D011247:Pregnancy; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014500:Uracil Nucleotides / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D000345:Affinity Labels; D001894:Borohydrides; D014500:Uracil Nucleotides; D014539:Uridine Diphosphate Sugars; D014531:Uridine Diphosphate Galactose; D008345:Manganese; D005700:Galactosyltransferases