Electric Potential and pH Gradient Formation by Reconstituted ATPase Proteo-Liposomes from the Thermophilic Cyanobacterium Synechococcus 6716

Abstract

The proton-translocating ATPase complex has been the object of many studies to elucidate its energy-transducing function in the direction of both ATP synthesis and hydrolysis. By reconstitution in liposomes, mostly prepared from asolectin, a simple and functional model system can be obtained. Recently, a DCCD-sensitive H+-ATPase complex has been isolated from the thermophilic cyanobacterium Synechococcus 6716 (Lubberding et al., submitted) as a Mg2+-dependent heat-stable complex, which can only be activated by trypsin. This complex, reconstituted with liposomes from isolated Synechococcus lipids catalyzes DCCD-sensitive 32Pi-ATP exchange and ATP hydrolysis activities and generation of an uncoupler-sensitive transmembrane electric potential (Δψ) and a pH gradient (ΔpH) during ATP hydrolysis (Van Walraven et al., submitted). In this paper we describe the ATP-dependent membrane energization by the use of the surface charge density probe TDACMA, the electric potential-sensitive dye oxonol VI and natural carotenoids and the pH indicators neutral red and cresol red, trapped inside the vesicles, and their sensitivity to the iono-phores valinomycin and nigericin.