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-transfe rase) by scintillation proximity assay (SPA) was developed. The assay quantitates the radioactivity incorporated from H-3-labeled UDP-GalNAc into a biotin-labeled acceptor peptide, as measured after adsorption of the acceptor peptide to avidin-coated SPA beads. The acceptor pepti de, PPASTSAPG (Elhammer et al. (1993) J. Biol. Chem. 268, 10029-10038) was conjugated to biotin using a di-beta-alanine spacer arm. The conj ugated peptide reacted readily with the enzyme and it had an apparent K-m comparable to that of the parent peptide. Using a reaction mixture consisting of 4 mg of SPA beads, 17 mu M acceptor, 0.5 mu M nucleotid e sugar, and 7.5 U/ml enzyme, the time dependence of product formation obeyed Michaelis-Menten-type kinetics throughout the full course of t he reaction-until exhaustion of the donor substrate-and the beginning portion of the reaction was sufficiently linear for calculating accura te initial rates, Analysis of the time dependency yielded an apparent K-m of 0.38+/-0.12 mu M for UDP-GalNAc. The assay is conveniently carr ied out in 96-well microtiter plates; it is ideally suited for assayin g large numbers of samples and for screening large collections of chem icals for competitive inhibitors. (C) 1996 Academic Press, Inc.