Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Volume 1797, Issue 8, Pages 1540-1545Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbabio.2010.03.021
Keywords
Steady-state kinetics; ATP hydrolysis; ADP inhibition; Thermodynamic efficiency; Pig kidney; Shark rectal gland
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Funding
- Royal North Shore Hospital, Sydney
- Deutscher Akademischer Austauschdienst (DAAD)
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Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na+,K+-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of -51 (+/- 1) kJ mol(-1). This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na+ and K+ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis-Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K-m and K-I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays. (C) 2010 Elsevier B.V. All rights reserved.
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