Subunit stoichiometry and interaction of F1-ATPase of beef heart mitochondria

By incubating soluble F1 of the beef heart mitochondria with radio active 7-chloro-4-nitro-2,1,3-benzoxadiazole (14C)-NBD-C1 in the dark, a labeled F1 with NBD/F1 molar ratio of 1:1 (0.995:1) was obtained, but 98% of the ATPase activity was inhibited. The inactivated ATPase activity of the NBD-labeled F1 may be restored by DTT, suggesting that an essential tyrosine residue of F1 (O-NBD-F1) may be labeled. By exposure of O-NBD-F1 with 0.83 NBD label per F1 molecule and a stable 82.5% inhibition of the ATPase activity even in the presence of DTT. Urea PAGE analysis clearly showed that a 63.4% of the total radio activity was associated with the beta-subunit. In the beta-fraction from N-NBD-F1 only a 19.90-20% of the labeling was detected. It is calculated that F1 of the beef heart mitochondria contains 3 (2.63-2.64) beta-subunits. The results obtained are discussed in connection with the subunit-subunit and site-site interactions and three-site model for F1 catalysis proposal by Boyer and his group.