Approaching determination of H+-ATPase H+/ATP stoichiometry

Sammanfattning: The plant plasma membrane H+-ATPase was activated by the binding of a 14-3-3 protein. This complex can be stabilized by the fungal toxin - fusicoccin. During this activation H+/ATP stoichiometry changed (1). This work presents approaching to find the new stoichiometry. The main part presents looking for the best preparation of plasma membrane vesicles for measurements. A new pH indicator – Glu3 – was trapped inside vesicles in a few different ways such as Brij 58 treatment or sonication. Membranes were washed by ultracentrifugations or gel chromatography. The tight of prepared vesicles was examined by acid titration. The best result was achieved by sonication and gel chromatography. Brij 58 also affects the size of plasma membrane vesicles what was demonstrated. The average diameter decreases with increasing concentration of detergent.