Determination of beta 1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection

We have developed a nonradioactive method to assay UDP-Gal:beta-D-GlcNAc be ta 1,4-galactosyltransferase (beta 4GalT-I) enzymatic activity. Capillary e lectrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-c ontaining substrate peptides and galactose-capped product peptides, while a t the same time allowing a level of detection in the low attomole range (10 (-18)). The addition of 2 mM hexamethylene diamine to the berate-based capi llary electrophoretic buffer modulated the electroosmotic how resulting in optimum separation of the glycopeptide product from reactant. beta 4GalT-I activity was dependent upon the addition of both manganese and UDP-galactos e, Using this assay, we show that two beta 4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translat ion system. The high sensitivity of product detection by CE-LIF in combinat ion with in vitro transcription-translation is applicable to the facile det ermination of the enzymatic activity of other newly cloned glycosyltransfer ases. (C) 1999 Academic Press.