Plant leaf cells constitute a unique system in which both mitochondria and chloroplasts are responsible for energy conversion. In both organelles, ATP synthase is responsible for the synthesis of ATP from ADP and Pi in a process coupled to an electron transfer chain.

ATP synthase is a membrane-bound enzyme with a highly conserved structure in mitochondria and chloroplasts as well as in bacteria. The enzyme consists of two morphologically and functionally distinct domains, the catalytic Fi part and the H+-translocating F0 part. Although the chloroplast enzyme has been isolated and extensively characterized, difficulties associated with the purification of plant mitochondria contributed to the poor understanding of the plant mitochondrial ATP synthase. In the present study we have developed a large-scale purification procedure for spinach mitochondria almost free from thylakoid contaminants and, for the first time, purified and characterized the structural and catalytic properties of the plant mitochondrial ATP synthase.

Isolation and purification of the spinach mitochondrial ATP synthase was achieved by a procedure involving 3-[(3-cholamidopropyl) dimethylammonio]-l-propanesulfonate (CHAPS) extraction of the enzyme from the inner mitochondrial membrane followed by density gradient centrifugation.

The isolated complex consists of twelve polypeptides. Based on N-terminal amino acid sequence analysis and Western blotting using monospecific antibodies raised against proteins characterized in other sources, we identified five polypeptides of molecular masses 54 kDa (a,ß), 33 kDa (y), 21 kDa (8) and 9.5 kDa (e) as being the components of the catalytic Fi sector of the enzyme, and the remaining polypeptides of molecular masses of 28 kDa Qj), 23 kDa (OSCP), 18.5 kDa (a), 15 kDa (Fg), 10.5 kDa (lFi), 9.5 kDa (c) and 8.5 kDa (unknown) as the components of the F0 sector of the complex.

Reconstitution experiments using Fi-depleted potato submitochondrial particles and the isolated potato subunits were used to study the role of the individual subunits of the complex. The Fj depleted particles were reconstitutively active. The 27 kDa and 23 kDa proteins which were found correspond to F0 subunit b. and OSCP, respectively, were also shown to be important for correct organization of the complex after reconstitution.

A small, 9.5 kDa, loosely bound protein was identified as an endogenous ATPase inhibitor protein (lFi) and was found to play an important role in the regulation of ATPase activity of potato mitochondria. By the use of a simple procedure, this protein was for the first time purified and characterized from a plant source. Comparison of the potato inhibitor protein with those from other sources revealed the close structural and functional relationship between the plant and the yeast inhibitors.

The FqFi complex in both membrane-bound and solublized forms catalyzes oligomycin-sensitive hydrolysis of the purine nucleotides in the order GTP > FTP > ATP. Hydrolysis of ATP, but not of other nucleotides was stimulated several fold in the presence of activators, such as anions and detergents. On the basis of these results, we classified the plant mitochondrial enzyme as a "similatent” ATPase.

Translocation of the transmembrane proton gradient set up by respiration through the ATPase complex was measured in potato submitochondrial particles before and after depletion of Fi. Kinetic analysis of inhibition of the H+-translocation by oligomycin in both types of membranes showed an initial fast phase. Almost complete inhibition with 1 mol oligomycin per mol ATP synthase was observed in both types of particles, indicating that removal of Fi from the particles does not alter the kinetics of proton translocation through F0 and the interaction of F0 with oligomycin.