Equilibrium binding of nucleotides to beef heart mitochondrial adenosine triphosphatase

The proton-ATPase of bacteria and mitochondria

“…In the membrane-bound enzyme formation of AfiH + seemed to sharply increase AMPPNP release. In fact, during oxidative phosphorylation, levels of AMPPNP up to 1.1 mM do not appreciably inhibit (Penefsky, 1974;Pederi0 The exact agreement between this value and the value deduced earlier by Hilborn and Hammes (1973) for equilibrium binding of ADP to soluble F~ should be noted. Because the value of 0.28 g~ was far below the measured apparent K~ ADP, Hilborn and Hammes suggested that ADP was not binding at the catalytic site, but possibly to a regulatory site.…”

Electron Transport Phosphorylation

“…In earlier studies they had observed that the competitive inhibition of the ATPase reaction exerted by the product ADP is much stronger in the soluble than in the membrane-bound enzyme (147), with a K~ at 8.0 of 30 t~M as determined with the soluble.and 80 vM with the membrane-bound ATPase. The similarity between the K i and the dissociation constant for ADP at the loose binding site suggests that this is the catalytic site for' the ATPase reaction (145). This conclusion is supported also by the fact that the hydrolysis reaction not markedly inhibited when the tight site is occupied by ADP or IDP (145, 12~7).…”

“…The observed distinction between a tight and a loose binding site (145) in solubilized beef heart mitochondrial 1 f or ADP, in the presence of Mg 2+ ion, with dissociation constants at pH 8.0 of 0.28 /xM and 47 #M, respectively, is of great interest. The only site observed in F 1 from rat liver mitochondria (143) gave a dissociation constant of 0.94/~M and was thus more of the tight type.…”

“…v I VROM MITOCIaONDRIA In mitochondrial coupling factors originating from beef heart or rat liver, the existence of one (143) or two (144,145) binding sites for (and none for ATP or AMP) has been reported from several laboratories. Very recently the coupling factor F 1 isolated from beef heart was found by Harris et al (146) to contain as many as 5 moles of tightly bound adenine nucleotide per mole of enzyme.…”

“…Hilborn & Hammes (145) found that the tight coupling site of isolated beef heart F 1 may be missing or have a reduced binding affinity when the ATPase is attached to its native mitochondrial membrane. In earlier studies they had observed that the competitive inhibition of the ATPase reaction exerted by the product ADP is much stronger in the soluble than in the membrane-bound enzyme (147), with a K~ at 8.0 of 30 t~M as determined with the soluble.and 80 vM with the membrane-bound ATPase.…”