In vivo analysis of glycogen Biosynthetic Pathway in Anabaena sp. and E. coli

RIUS, SP; IGLESIAS AA; GÓMEZ-CASATI DF

Bariloche

Congreso; XXXIX Reunión Nacional. Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2004

SAIB

Cyanobacterial and E. coli cells were permeabilized using a mixture of toluene:ethanol or toluene, respectively. Permeabilization was confirmed after addition of fluorescein diacetate and microscopic visualization. After the incubation of the treated cells with ADPGlc PPase substrates, ATP and  [14C]Glc1P; or glycogen synthase substrate, [14C]ADPGlc,  labelled alpha-1,4-glucan was recovered. Addition of external non  radioactive ADPGlc in the incubation media with ATP and  [14C]Glc1P did not affect the [14C]Glc incorporation into the  glucan. Moreover, there was only 40% of the total incorporation  of [14C]ADPGlc when the cells were in the presence of ADPGlc  PPase non-labelled substrates. Structural data from Blue native PAGE, western blot analysis and co-immunoprecipitation, suggest  the existence of protein-protein interactions between the enzymes involved in glycogen biosynthesis in Anabaena and E. coli. Results suggest a direct transfer of ADPGlc between ADPGlc PPase and glycogen synthase (metabolite channeling). This is a process by which a metabolite is directly transferred from one enzyme active site to the next without being released free into solution. The direct channeling of an intermediate between enzymes catalyzing consecutive reactions in a biochemical pathway offers the possibility of an efficient and exclusive metabolite delivery. This mechanism may allow to maintain a high flux of substrates in the glycogen biosynthetic pathway under different physiological conditions.