CHARACTERIZATION AND PREVENTION OF THE PROTEOLYTIC CLEAVAGE OF PLANT PHOSPHO-ENOLPYRUVATE CARBOXYKINASE.

MARTÍN, M.; PODESTÁ, F. E.

Bariloche, Argentina

Congreso; XXXIX Reunión anual de la SAIB; 2003

In plants, phosphoenolpyruvate carboxykinase (PEPCK) catalyses a key step in the gluconeogenesis during germination of fat storing seeds, plays an important role in photosynthetic carbon assimilation in some CAM and C4 plants, as well as in the CO2–concentrating mechanism of certain algae. Plants PEPCKs have unique, species-specific, N-terminal extensions that are rapidly lost by proteolysis during preparation of cell extracts. Being the site of reversible phosphorylation, proteolytic cleavage of these extensions hinders the study of the regulation of PEPCK activity. The aim of this study was to find proper conditions to extract and purify native PEPCK from endosperm of 5 days old Ricinus communis germinating seeds. PEPCK presented a subunit molecular mass of 80 kDa in extracts obtained under denaturating conditions when analyzed by western blot using affinity-purified antibodies against truncated PEPCK from pineapple leaves. Studying the time course state of PEPCK subunit in extracts made at different pH values with and without DTT, we concluded that a cysteine endopeptidase might be responsible for the degradation of PEPCK to a truncated subunit form of 66 kDa. A wide range of protease inhibitors was tested but none was completely useful to prevent proteolysis. A combination of high pH and protease inhibitors was found to be most effective to prevent proteolytic cleavage and maintain activity of PEPCK.