Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria

Abstract

AbstractA simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled K-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Keywords: Glycogen; Starch synthesis; Cyanobacteria; Anabaena PCC 7120; Glycogen synthase; Permeabilized cell ; Metabolic £ux measurement;ADP-glucose pyrophosphorylase