Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).
Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D002417:Cattle; D004579:Electron Transport; D007700:Kinetics; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009206:Myocardium; D010085:Oxidative Phosphorylation; D011485:Protein Binding; D014357:Trypsin
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases