The GALE reaction catalyzes the reversible conversion of uridine diphosphate galactose (UDPGal) to uridine diphosphate glucose (UDPGlc).
The RPMI media, L-glutamine, and glycine (culture grade) were from Fisher Scientific; fetal bovine serum and HEPES from Invitrogen; and UDPGal ([greater than or equal to] 97%), UDPGlc ([greater than or equal to] 98%), and all other chemicals and reagents from Sigma-Aldrich.
The calibrators had 2 variables: one was the total concentration of UDPGal and UDPGlc (UDPGal and UDPGlc) and the other was the percentage of UDPGal and UDPGlc that was accounted for by UDPGal (Table 1).
We evaluated recovery of the assay by spiking a known amount of UDPGlc into reaction mixtures in which the enzyme substrate, UDPGal, was added after the reaction mixture was inactivated by boiling the samples for 3 min.
Fragmentation of UDPGal and UDPGlc showed almost identical spectra.
To calculate the enzymatic product of UDPGlc in the reaction, we first needed to calculate the total amount of UDPGal and UDPGlc.
(1992) Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDPGlc
Glycine (culture grade) was purchased from Fisher Scientific, and uridine diphosphate glucose (UDPGlc) (disodium salt from Saccharomyces cerevisiae) and all other chemicals were obtained from Sigma.
Initially, 16 [micro]L each of the 0.5 mol/L glycine buffer (pH 8.7), 2.0 mmol/L UDPGlc, and 8.0 mmol/L [[sup.13]C6]-Gal-1-P solutions were added to a 1.5-mL Eppendorf tube.
In this experiment, GALT activities in the erythrocytes from 15 healthy individuals and 15 patients with GALT deficiency were determined by both the LC-MS/MS assay and a reference assay that measures GALT activity by using fluorescent measurement to determine the consumption of UDPGlc (5).
1D (565 > 323), which is a combination of UDPGlc and UDPGal, owing to the fact that they have the same retention time and fragmentation pattern.
Optimal substrate concentrations (of both [[sup.13][C.sub.6]]-Gal-1-P and UDPGlc) and linearity with time and protein concentration were determined.