Measurement of glycogen synthase activity in crude extracts by CE.
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 radioassay. 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 microA) in uncoated capillaries (53 cmx50 microm). Sample injection time was 30 s and nucleotides 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 to15 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.
Wilson
Christian A M
CA
Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla, Santiago, Chile.
Preller
Ana
A
Valenzuela
María A
MA
Ureta
Tito
T
D000818:Animals; D001001:Anura / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D006006:Glycogen Synthase / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D066298:In Vitro Techniques; D009865:Oocytes / Q000201:enzymology; D014530:Uridine Diphosphate / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D014530:Uridine Diphosphate; D006006:Glycogen Synthase; D014532:Uridine Diphosphate Glucose