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.
Baker
C A
CA
Pharmacia & Upjohn, Inc., 301 Henrietta Street, Kalamazoo, Michigan, 49001, USA.
Poorman
R A
RA
Kézdy
F J
FJ
Staples
D J
DJ
Smith
C W
CW
Elhammer
A P
AP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D007700:Kinetics; D008863:Microspheres; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis*; D010455:Peptides / Q000378:metabolism; D012588:Scintillation Counting; D019809:Streptavidin; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D001426:Bacterial Proteins; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides; D019809:Streptavidin; D000097763:Polypeptide N-acetylgalactosaminyltransferase