Determination of β1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection

D. M. Snow, J. H. Shaper, N. L. Shaper, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

We have developed a nonradioactive method to assay UDP-Gal:β-D- GlcNAcβ1,4-galactosyltransferase (β4GalT-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. β4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two β4GalT-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.

Original languageEnglish (US)
Pages (from-to)36-42
Number of pages7
JournalAnalytical Biochemistry
Volume271
Issue number1
DOIs
StatePublished - Jun 15 1999

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Galactosyltransferases
Capillary electrophoresis
Capillary Electrophoresis
Transcription
Assays
Lasers
Fluorescence
Uridine Diphosphate Galactose
Electroosmosis
Glycosyltransferases
Peptides
Borates
Uridine Diphosphate
Diamines
Glycopeptides
Fluorescein-5-isothiocyanate
Reticulocytes
Manganese
Galactose
Buffers

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Determination of β1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection. / Snow, D. M.; Shaper, J. H.; Shaper, N. L.; Hart, Gerald Warren.

In: Analytical Biochemistry, Vol. 271, No. 1, 15.06.1999, p. 36-42.

Research output: Contribution to journalArticle

Snow, D. M. ; Shaper, J. H. ; Shaper, N. L. ; Hart, Gerald Warren. / Determination of β1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection. In: Analytical Biochemistry. 1999 ; Vol. 271, No. 1. pp. 36-42.
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