| Abstract: | The H+-PPase and the H+-ATPase of the vacuolar membrane were separated during purification of tonoplast proteins of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Three membrane protein fractions prepared contained firstly, the H+-PPase protein without any subunits of the H+-ATPase, secondly, the H+-PPase protein with only minute traces of the intramembraneous 16 kDa c-subunit of the H+-ATPase, and thirdly, the H+-ATPase subunits without H+-PPase peptides as verified by SDS-PAGE. These three preparations were reconstituted into soybean (Glycine max L.)-phospholipid vesicles, and compared with proteoliposomes obtained by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze-fracture replicas prepared from these five different types of vesicles showed that there are two populations of intramembraneous particles, one with a diameter of 6.7-7.2 nm corresponding to the H+-PPase, and one with an average diameter of 9.1 nm belonging to the H+-ATPase. Thus, freeze-fracture electron microscopy allows one to visualize H+-PPase particles in addition to H+-ATPase particles in the tonoplast of Kalanchoë daigremontiana. |
