Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase.
MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D017830:Octoxynol; D010042:Ouabain / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D011092:Polyethylene Glycols; D008345:Manganese; D010042:Ouabain; D017830:Octoxynol; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium