A scintillation proximity assay for UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase

A rapid and simple method for quantitating the reaction product of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from 3H-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor peptide, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029-10038) was conjugated to biotin using a di-beta-alanine spacer arm. The conjugated peptide reacted readily with the enzyme and it had an apparent Km comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 microM acceptor, 0.5 microM nucleotide sugar, and 7.5 U/ml enzyme, the time dependence of product formation obeyed Michaelis-Menten-type kinetics throughout the full course of the reaction-until exhaustion of the donor substrate-and the beginning portion of the reaction was sufficiently linear for calculating accurate initial rates. Analysis of the time dependency yielded an apparent Km of 0.38 +/- 0.12 microM for UDP-GalNAc. The assay is conveniently carried out in 96-well microtiter plates; it is ideally suited for assaying large numbers of samples and for screening large collections of chemicals for competitive inhibitors.