کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1942842 1052632 2010 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Investigation of the enzymatic activity of the Na+,K+-ATPase via isothermal titration microcalorimetry
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک دانش گیاه شناسی
پیش نمایش صفحه اول مقاله
Investigation of the enzymatic activity of the Na+,K+-ATPase via isothermal titration microcalorimetry
چکیده انگلیسی

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 Km and KI 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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Bioenergetics - Volume 1797, Issue 8, August 2010, Pages 1540–1545
نویسندگان
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