Abstract
In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the Department of Physiology, UCLA School of Medicine and Center for Ulcer Research and Education, Membrane Biology Laboratory, Veterans Administration/ WadsworthHospital, Los Angeles, California90073
The inabilityof the Ca-EP to dephosphorylate in the Ca2+,K+-ATPaseactivity was only0.7% of the Mc,K+-ATPase, onstrates a second action of the divalent cation: that indicating that calcium could partially substitute for the typeof divalent cation whicohccupies the catalytic
Proton transport the divalent cationsite of the phosphoenzyme and the measured by acridine orange uptake wans ot detected occupation of this site bycalcium causes slowerphosin thepresence of Ca2+and K+.In thepresence of M S + phoenzyme kinetics
Summary
Solic K+,EzPdephosphorylates to form EzK+at a rate thatis faster thanenzyme turnover [4].Under these conditions the rate-limiting step is assumed to occur after dephosphorylation but to precede phosphorylation. The dephosphorylation reaction is thought to require Mg2+ because phosphorylation. Reagents [ Y - ~ ~ P ] AwTaPs purchased from Amersham Corp. "vanadium free" &sodium ATP was obtained from Sigma. All other reagents were a t least of reagent grade. From PO, and "0-PO4 medium exchange require M P [5,6]. The EzK+form converts to the EIK+form of the enzyme to release K+ into the cytosol, rebind H+, and toinitiate a new catalytic cycle with the binding of ATP
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