(Na+ + K+)-ATPase : phosphorylation-dependent cross-linking of the alpha-subunits in the presence of Ca2+ and o-phenanthroline.
In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003300:Copper / Q000494:pharmacology*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis*; D007679:Kidney Medulla / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D014544:Uridine Triphosphate / Q000494:pharmacology
D004792:Enzyme Precursors; D010618:Phenanthrolines; D003300:Copper; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D014544:Uridine Triphosphate