A solid-phase assay for beta-1,4-galactosyltransferase activity in human serum using recombinant aequorin.
We have developed a sensitive and rapid solid-phase assay for the serum enzyme UDPGal:beta-D-GlcNAc beta-1,4-galactosyltransferase (beta 1,4-GT) (EC 2.4.1.38) that employs the recombinant bioluminescent protein aequorin as the enzyme label for product detection. The substrate for beta 1,4-GT is a neoglycoprotein, bovine serum albumin containing covalently attached GlcNAc residues (GlcNAc-BSA), and it was immobilized by adsorption in microtiter plate wells. Serum samples were added to each well along with saturating levels of UDPGal and Mn2+. Galactosylation of the neoglycoprotein acceptor by the serum beta 1,4-GT produces the N-acetyllactosamine derivative Gal beta 1, 4GlcNAc-BSA. The product formed is quantified by adding the biotinylated plant lectin Ricinus communis agglutinin-I, which binds specifically to N-acetyllactosamine, followed by the addition of streptavidin and the biotinylated aequorin. Aequorin produces a flash of light in response to Ca2+ and is detectable to 10(-19) mol in a luminometer. Using this assay, the beta 1,4-GT activity in human serum and the activity of a semipurified beta 1,4-GT are linear with time and serum concentration over a wide range. The reaction is dependent on UDPGal and Mn2+, is highly reproducible with a low background, and can be performed in a few hours. Assays employing aequorin have a wider range of linearity than those employing horseradish peroxidase as an enzyme label. These results demonstrate that the assay for beta 1,4-GT is useful for determining activity in heterogeneous samples and also demonstrate the utility of the recombinant protein aequorin for solid-phase assay methods.
Zatta
P F
PF
Department of Biochemistry, University of Georgia, Athens 30602.
Nyame
K
K
Cormier
M J
MJ
Mattox
S A
SA
Prieto
P A
PA
Smith
D F
DF
Cummings
R D
RD
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000331:Aequorin / Q000097:blood*; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002240:Carbohydrate Sequence; D002417:Cattle; D005700:Galactosyltransferases / Q000097:blood*; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008832:Microchemistry / Q000379:methods; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D037121:Plant Lectins; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
D037102:Lectins; C050080:N-acetylglucosamine-bovine serum albumin conjugate; D009844:Oligosaccharides; D037121:Plant Lectins; D011994:Recombinant Proteins; C017707:Ricinus communis agglutinin-1; D012710:Serum Albumin, Bovine; D000331:Aequorin; D001710:Biotin; D005700:Galactosyltransferases; D000117:Acetylglucosamine