A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-β-D-maltoside

Academic Article

Abstract

  • Glycogenin is a self-glycosylating protein that catalyzes early glucosyl transfer steps in the biosynthesis of glycogen. In currently used assays of glycogenin activity, the enzyme is incubated with radioactive UDP-glucose, and the labeled reaction product is then isolated by precipitation with trichloroacetic acid. A new assay is reported here which is based on the observation that glycogenin is not only self-glycosylating but may also use exogenous alkyl maltosides as substrates. After incubation of the enzyme with n-dodecyl-β-D-maltoside and UDP-[3H]glucose, the radioactivity in the resultant n-dodecyl-β-D-[3H]maltotrioside is determined by any one of the following three procedures, which all rely on the hydrophobic properties conferred on the reaction product by the alkyl aglycone: (i) adsorption of the product to a Sep-Pak C18 cartridge and elution with 70% ethanol; (ii) biphasic liquid scintillation counting in ScintiLene/25% isoamyl alcohol, without isolation of the product, and (iii) precipitation with trichloroacetic acid in the presence of carrier protein. The Sep-Pak C18 procedure has the advantage that it allows essentially quantitative isolation of the reaction product, while, under the conditions chosen, only about 50% of the product is precipitated by trichloroacetic acid. For most applications, however, biphasic liquid scintillation counting is the method of choice, since close to 90% of the labeled product is extracted into the organic phase and can be counted directly without interference from the labeled nucleotide sugar which remains in the aqueous phase. In crude enzyme extracts, where nucleotide sugar pyrophosphatase and alkaline phosphatase activities are high, inclusion of 1 mM β-NAD and unlabeled UDP-glucose at a final concentration of 10 μM in the reaction mixtures minimizes the apparent breakdown of the radioactive nucleotide sugar, which results in a high background due to extraction of [3H]glucose into the organic phase. The biphasic procedure is potentially applicable to the assay of any glycosyltransferase, for which a suitable hydrophobic acceptor is available. © 1994 Academic Press, Inc.
  • Authors

    Digital Object Identifier (doi)

    Author List

  • Manzella SM; Rodéen L; Meezan E
  • Start Page

  • 383
  • End Page

  • 391
  • Volume

  • 216
  • Issue

  • 2