Abstract

Glycogen synthase catalyzes the incorporation of UDP-glucose into glycogen. The activity of the enzyme is usually measured either by a spectrophotometric method or by a radio-assay. The first one is not suitable because of the difficulties regarding the use of coupled enzymes in crude extracts, while the second is a time-consuming method involving glycogen isolation and manipulation of radioactivity. We have used a CZE technique as a novel approach to measure glycogen synthase activity. The separations were performed at 22 kV (36 μA) in uncoated capillaries (53 cm × 50 μm). Sample injection time was 30 s and nudeotides were monitored at 254 nm. Best resolution was achieved in 20 mM tetraborate buffer, pH 9.2. Curves of absorbance as a function of UDP and UDP-glucose concentration were linear. Enzyme activity in oocyte extracts was linear with respect to time (up to 15 min) and enzyme concentration. The K m app. for UDP-glucose was 0.87 mM, a value identical to the one reported using the radioassay. CZE enables easy quantitation of compounds, high sensitivity; and automation of the process. Small sample sizes are required, interferences by auxiliary enzymes and manipulation of radioactivity are avoided, and analysis time is significantly diminished. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.