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

Abstract 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 3 H-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-β-alanine spacer arm. The conjugated 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 μ M acceptor, 0.5 μ M 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 K m of 0.38 ± 0.12 μ M 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.