F1-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F1 contains about 2 mol of Mg2+/mol of F1, 1 mol of which can be removed or exchanged. The remaining mole of Mg2+ per mole of F1 remains very tightly associated with F1 and is recovered in the αγ fraction after cold denaturation. Rat liver F1also contains as isolated a nearly equivalent amount of nucleotide (~ 1.7 mol/mol of F1) which is readily removed by incubation at room temperature followed by column centrifugation. The “2 Mg2+enzyme” binds almost 3 mol of 5’-adenylyl imidodiphosphate (AMP-PNP)/mol of F1in the presence or absence of added divalent cation. When divalent cation is present as Co2+, an equivalent activator to Mg2+ in the ATPase reaction, 1 mol of F1 binds 3 mol of both AMP-PNP and Co2+. Under these conditions, the very tight Mg2+ site remains loaded, the exchangeable Mg2+ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F1 contains only a single readily detectable ADP binding site in the presence or absence of divalent cation. The covalent labeling agent 7-chloro-4-nitro-2, l, 3-benzoxadiazole prevents binding of all three AMP-PNP molecules and two of the three Co2+ molecules and reduces bound ADP by about 50%. Carboxylate labeling agents and a variety of other covalent labeling agents are without effect on Co2+ binding to F1. These results are best interpreted within the framework of the asymmetric structure of the rat liver F1complex which contains only a single copy of the smaller subunits (γδ∊) per three α(β pairs. It is suggested that the ADP site, the tight Mg2+site, and the exchangeable Mg2+ site (capable of forming an Mg-ATP complex) lie at the asymmetric center of F1i.e., on an αβ pair “tagged” by one or more of the smaller subunits. In contrast, the two additional ATP metal binding sites are considered to lie on “naked” or “pure” αβ subunit pairs. Three current models for the function of F1 are discussed with respect to their capacity to accommodate the ligand binding data obtained for the rat liver enzyme.
ASJC Scopus subject areas