The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations.

摘要: Abstract In order to determine the role of divalent cations in reaction mechanism H+,K+-ATPase, we have substituted calcium for magnesium, which is required by H+,K+-ATPase phosphorylation from ATP and PO4. Calcium was chosen over other assayed (barium manganese) because absence activated hydrolysis, generated sufficiently high levels phosphoenzyme (573 +/- 51 pmol.mg-1) [gamma-32P]ATP study dephosphorylation, inhibited K+-stimulated hydrolysis. The Ca2+-ATPase activity 40% basal Mg2+-ATPase activity. However, Ca2+,K+-ATPase (minus Ca2+ activity) only 0.7% Mg2+,K+-ATPase, indicating that could partially substitute Mg2+ activating hydrolysis but not K+ stimulation Approximately 0.1 mM 50% or Mg2+,K+-ATPase activities. Inhibition competitive with respect K+. Mg2+,K+ p-nitrophenyl phosphatase an apparent Ki 0.27 mM. Proton transport measured acridine orange uptake detected presence K+, proton affinity similar inhibition Mg2+, K+-ATPase site on exterior vesicle. These results suggest activates turnover inhibits binding at a cytosolic cation site. pseudo-first rate constant formation 5 microM least 22 times slower (0.015 s-1) than magnesium (greater 0.310 s-1). Ca.EP (phosphoenzyme formed Ca2+) dephosphorylated four five more slowly Mg.EP Mg2+) 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) 250 ATP. 10% sensitive 100 KCl chase compared greater 85% Mg.EP. By comparing transient kinetics (Mg.EP) (Ca.EP), found two actions dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)