Functional Characterization of Human Glycogen Branching Enzyme obtained in Sf9 cells

ISSOGLIO, FEDERICO M.; CARRIZO, MARÍA E.; CURTINO, JUAN A.

Carlos Paz, Córdoba

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2013

Sociedad Argentina de Biofísica (SAB)

Glucose is the principal source of energy for most cells. In mammals, glucose is stored as glycogen, the branched polymer formed by linear α-1,4-oligoglucan chains linked by α-1,6-glucosidic bonds. Three enzymes are mainly responsible for the de novo biosynthesis of glycogen. First, glycogenin autoglucosylation produces a protein-bound oligoglucan that serves as primer for the other two enzymes, glycogen synthase elongating the chains, and the glycogen branching enzyme catalyzing the cleavage of a linear segment and transferring it to the 6-position of a non-terminal glucosyl unit. Glycogen branching enzyme (GBE) is the least studied of the three enzymes. It has been isolated and characterized in some bacteria, rabbit and rat. In this study we report the production of recombinant human GBE in insect cells using the baculovirus expression system, and the activity analysis of the recombinant enzyme. Using amylose as a substrate, the branches introduced by GBE are analyzed after degradation with isoamylase. The new reducing oligosaccharides generated are subjected to fluorophore-assisted carbohydrate electrophoresis (FACE)1, where saccharides are labeled quantitatively with a fluorophore and then separated by polyacrilamide gel electrophoresis (PAGE). Using this method we could determine the length preference of the transferred oligosaccharide chain, and measure GBE activity quantifying the specific reaction product. 1. Starr et al. (1996) J. Chromatogr. A, 295.