Studies on (K+ + H+)-ATPase III. Binding of adenylyl imidodiphosphate.
1. Adenylyl imidodiphosphate (AMPPNP) binds to (K+ + H+)-ATPase from pig gastric mucosa with a dissociation constant (Kd) of 50 microM for the AMPPNP-enzyme complex. 2. Monovalent cations reduce the amount of AMPPNP bound in the following order of effectiveness Tl+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+, Li+, choline+. 3. AMPPNP binding to the enzyme has a pH optimum at pH 7.0--7.5 in the absence of added ions, which is shifted to pH 8 upon addition of MgCl2. 4. Cyclodiaminotetraacetic acid (CDTA, Tris salt) inhibits binding of AMPPNP. This inhibition is not due to chelation of Mg2+. It may be due to direct binding of CDTA to the enzyme or to removal of stabilizing cations other than Mg2+. 5. Binding curves determined in the presence of various concentrations of Mg2+ show that at low Mg2+ concentrations (less than 0.5 mM), the apparent number of binding sites is reduced, while at higher Mg2+ concentrations (greater than or equal to 0.5 mM), the binding of AMPPNP is inhibited in a competitive way. 6. From these observations it is concluded that the enzyme has two binding sites for AMPPNP and only one for Mg-AMPPNP (or two with strong anti-cooperativity), and that Mg2+ inhibits binding of Mg-AMPPNP. This finding is interpreted in terms of a model involving a dimeric form of the enzyme.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D000818:Animals; D002414:Cations, Monovalent / Q000494:pharmacology; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005753:Gastric Mucosa / Q000187:drug effects / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D013552:Swine
D002414:Cations, Monovalent; D000266:Adenylyl Imidodiphosphate; C005390:CDTA; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D008274:Magnesium