Topological localization of proteolytic sites of sodium and potassium ion-activated adenosine triphosphatase

Biochemistry ◽  
1983 ◽  
Vol 22 (14) ◽  
pp. 3405-3410 ◽  
Author(s):  
Gilbert Chin ◽  
Michael Forgac
Keyword(s):  
Adenosine Triphosphatase ◽  
Potassium Ion ◽  
Topological Localization ◽  
Sodium And Potassium

scholarly journals Equilibrium of Phosphointermediates of Sodium and Potassium Ion Transport Adenosine Triphosphatase

1997 ◽  
Vol 109 (5) ◽  
pp. 537-554 ◽  
Author(s):  
Kuniaki Suzuki ◽  
Robert L. Post
Keyword(s):  
Ion Transport ◽  
Rate Constants ◽  
Adenosine Triphosphatase ◽  
Reaction System ◽  
Potassium Ion ◽  
Hydrolysis Rate ◽  
Decay Kinetics ◽  
Potassium Ion Transport ◽  
Pool Model ◽  
Sodium And Potassium

Sodium and potassium ion transport adenosine triphosphatase accepts and donates a phosphate group in the course of its reaction sequence. The phosphorylated enzyme has two principal reactive states, E1P and E2P. E1P is formed reversibly from ATP in the presence of Na+ and is precursor to E2P, which equilibrates with Pi in the presence of K+. We studied equilibrium between these states at 4°C and the effect of Na+ on it. To optimize the reaction system we used a Hofmeister effect, replacing the usual anion, chloride, with a chaotropic anion, usually nitrate. We phosphorylated enzyme from canine kidney with [32P]ATP. We estimated interconversion rate constants for the reaction E1P ⇌ E2P and their ratio. To estimate rate constants we terminated phosphorylation and observed decay kinetics. We observed E1P or E2P selectively by adding K+ or ADP respectively. K+ dephosphorylates E2P leaving E1P as observable species; ADP dephosphorylates E1P leaving E2P as observable species. We fitted a 2-pool model comprising two reactive species or a twin 2-pool model, comprising a pair of independent 2-pool models, to the data and obtained interconversion and hydrolysis rate constants for each state. Replacing Na+ with Tris+ or lysine+ did not change the ratio of interconversion rate constants between E1P and E2P. Thus Na+ binds about equally strongly to E1P and E2P. This conclusion is consistent with a model of Pedemonte (1988. J. Theor. Biol. 134:165–182.). We found that Na+ affected another equilibrium, that of transphosphorylation between ATP·dephosphoenzyme and ADP·E1P. We used the reactions and model of Pickart and Jencks (1982. J. Biol. Chem. 257:5319–5322.) to generate and fit data. Decreasing the concentration of Na+ 10-fold shifted the equilibrium constant 10-fold favoring ADP·E1P over ATP·dephosphoenzyme. Thus Na+ can dissociate from E1P·Na3. Furthermore, we found two characteristics of Hofmeister effects on this enzyme.

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