VISUALIZATION BY FREEZE-FRACTURE ELECTRON-MICROSCOPY OF INTRAMEMBRANOUS PARTICLES CORRESPONDING TO THE TONOPLAST H-PYROPHOSPHATASE AND H+-ATPASE OF KALANCHOE-DAIGREMONTIANA HAMET ET PERRIER DE LA BATHIE()

The H+-PPase and the H+-ATPase of the vacuolar membrane were separated during purification of tonoplast proteins of Kalanchoe daigremontiana Hamet et Perrier de la Bathie. Three membrane protein fractions prepa red contained firstly, the H+-PPase protein without any subunits of th e 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-PA GE. These three preparations were reconstituted into soybean (Glycine max L.)-phospholipid vesicles, and compared with proteoliposomes obtai ned by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze-fracture replica s prepared from these five different types of vesicles showed that the re are two populations of intramembraneous particles, one with a diame ter of 6.7-7.2 nm corresponding to the H+-PPase, and one with an avera ge diameter of 9.1 nm belonging to the H+-ATPase. Thus, freeze-fractur e electron microscopy allows one to visualize H+-PPase particles in ad dition to H+-ATPase particles in the tonoplast of Kalanchoe daigremont iana.