Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria.
A 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 alpha-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.
Gómez Casati
D F
DF
Instituto Tecnológico de Chascomús (IIB-INTECH, CONICET), Camino Circunv. Laguna km 6, Casilla de Correo 164, 7130, Chascomús, Argentina.
Aon
M A
MA
Cortassa
S
S
Iglesias
A A
AA
D017033:Anabaena / Q000378:metabolism*; D002250:Carbon Radioisotopes / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D005936:Glucans / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis*; D006006:Glycogen Synthase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D014050:Toluene / Q000494:pharmacology
D002250:Carbon Radioisotopes; D005936:Glucans; D014050:Toluene; D000431:Ethanol; D006003:Glycogen; C040088:1,4-glucan; D006006:Glycogen Synthase