Ligand binding studies of the F1 moiety of rat liver ATP synthase: implications about the enzyme's structure and mechanism.
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 alpha gamma fraction after cold denaturation. Rat liver F1 also contains as isolated a nearly equivalent amount of nucleotide (approximately 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 F1 in 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D003035:Cobalt / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008930:Mitochondria, Liver / Q000201:enzymology*; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012265:Ribonucleotides / Q000378:metabolism
D008024:Ligands; D046911:Macromolecular Substances; D012265:Ribonucleotides; D000266:Adenylyl Imidodiphosphate; D003035:Cobalt; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; C018021:cobaltous chloride