Sodium ion dependence of inhibition of the Na +-translocating F 1F 0-ATPase from Acetobacterium woodii. Probing the site(s) involved in ion transport

Abstract

The Na +-translocating F 1F 0-ATPase of Acetobacterium woodii was stimulated not only by Na + but also by Li + and was protected by Na + or Li + from inactivation by N, N′-dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES) and tributyltin (TBSn) but not N-ethylmaleimide (NEM) or azide. The amount of Na + required for half-maximal protection from DCCD inhibition corresponded to the apparent K m for Na + of ATP hydrolysis. The inhibition by the amiloride derivatives hexamethylene-amiloride (HMA), ethylisopropylamiloride (EIPA), N-10-benzyl-amiloride (benzamil) and N-10-phenamil-amiloride (phenamil) could be relieved by Na + to various degrees. EIPA and HMA effectively protected the ATPase from DCCD inactivation, whereas the protection by benzamil and phenamil was only marginal indicating that the unsubstituted guanidinium group is essential for maximal protection from DCCD inactivation. These results indicate that the amiloride derivatives and Na + or DCCD compete for a common binding site. Chemical modification of histidine, arginine, aspartate or glutamate residues of the F 1F 0, complex resulted in an inhibition of ATP hydrolysis, indicating an essential function of these residues in the catalytic mechanism but this inhibition could not be relieved by Na +.