Intracellular flux measurements of ADPglucose pyrophosphorylase and glycogen in cyanobacteria

A.A. IGLESIAS, D.F. GÓMEZ CASATI

San Diego, California, USA

Congreso; Annual Meeting of the American Society of Plant Biologists (ASPB); 2000

ASPB

Cyanobacteria from the genus �Anabaena� sp., strain PCC7120, was permeabilized with a mixture of toluene:ethanol. Permeabilization was verified by loading with fluorescein diacetate and microscopic visualization. After excitation for fluorescein, non-permeabilized, viable, cells showed a green fluorescence whereas permeabilized cells exhibited only the intrinsic red fluorescence. When permeabilized cells were incubated with ADPglucose pyrophosphorylase (AGPase) substrates, ATP and radioactive glucose-P, labelled alpha-1,4 glucan was recovered, as could be judged through product sensitivity toward glucoamylase. AGPase exhibited low sensitivity to activation by its allosteric effector 3PGA in permeabilized �Anabaena� cells cultured under continuous illumination. However, when cells were preincubated one hour in the dark before permeabilization, AGPase became highly sensitive toward 3PGA. These observations are in agreement with the results obtained with a mathematical model of the dynamics of storage polysaccharide synthesis, according to which AGPase would be fully activated under continuous light. Modelling results, and experimental in vitro data obtained with AGPase under crowding conditions, show that the enzyme would function as a switch-like device for the polysaccharide synthesis during the light-dark transition. In fact, the enzyme was ultrasensitive with respect to 3PGA, and similar amplification factors ranging between 11- to 19-fold could be determined either experimentally or theoretically. The permeabilization system developed will allow us to investigate the ultrasensitivity phenomenon exhibited by AGPase under intracellular conditions.