Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.
We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.
Snow
D M
DM
School of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21207, USA.
Shaper
J H
JH
Shaper
N L
NL
Hart
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D005822:Genetic Vectors; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D066298:In Vitro Techniques; D007834:Lasers; D009237:N-Acetyllactosamine Synthase / Q000032:analysis* / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D006020:Glycopeptides; D009842:Oligopeptides; D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase