Human GALE also reversibly catalyzes the formation of UDP-N-acetylgalactosamine (UDP-GalNAc) from UDP-N-acetylglucosamine, which along with UDPGal and UDPGlc may be used in the synthesis of glycoproteins and/or glycolipids.
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).
0 mmol/L UDPGal solution, and 15 volumes of glycine buffer (0.
We examined the assay conditions for optimal substrate concentration by running the assay at different UDPGal concentrations (0.
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.
A product ion scan of UDPGal revealed 1 major ion of m/z 323 (negative ion of uridine monophosphate), which proved to be the strongest ion for [[sup.
The MS/MS transitions of 259 > 79 and 565 > 323 were employed for the detection of unlabeled Glu-1-P and UDPGal, respectively.
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.
In an independent study, a known amount of UDPGal in solution was spiked into a GALT assay sample after the reaction was quenched.
Although one radioactive assay has demonstrated the capability of measuring GALT activity of <5% of control values, this assay required overnight dialysis to remove small molecules in the hemolysate, which would lead to non-GALT-mediated formation of radioactive UDPGal (18).
The uridine nucleotide sugars, UDPGal and UDPGlc, which have identical HPLC retention time and MS/MS fragmentation, are shown as 1 single peak on the LC-MS/MS chromatogram.