Sauer:ATPase assays

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'''Principle.'''We use a system that couples ADP formation (from an ATPase of interest) with classical metabolic reactions to quantify ATP hydrolysis rates, as reference in Norby, JG (1988) ''Meth enzy'', '''156''', 116-9.  The basic principle can be summarized below:
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(1)  ATP <==> ADP + Pi
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(2)  ADP + Phosphenolpyruvate <==> ATP + Pyruvate
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(3)  Pyruvate + NADH + H+ <==> Lactate + NAD+
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where reaction (1) is catalyzed by your ATPase, reaction (2) by pyruvate kinase, and reaction (3) by lactate dehydrogenase.  Loss of NADH (as monitored by absorbance at 340 nm) is quantifiably proportional to ATP hydrolysis rates because this set of reactions begins with equilibria strongly favoring the forward direction and neither reactions (2) or (3) are rate-determining.
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'''Calculating the ATPase Rate.'''
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Total ATPase rate = (rate of A340 signal loss)/(pathlength*6.23mM^-1cm^-1)
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By applying the above formula, you'll get some rate expressed in ''mM per unit time''.  For the molar activity (i.e., rate per mole of enzyme) simply divide this rate by the enzyme concentration you used in the assay.  This will give you a hydrolysis rate of ''per unit time per enzyme''.
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'''Procedure'''

Revision as of 18:34, 4 November 2006

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Principle.We use a system that couples ADP formation (from an ATPase of interest) with classical metabolic reactions to quantify ATP hydrolysis rates, as reference in Norby, JG (1988) Meth enzy, 156, 116-9. The basic principle can be summarized below:


(1) ATP <==> ADP + Pi

(2) ADP + Phosphenolpyruvate <==> ATP + Pyruvate

(3) Pyruvate + NADH + H+ <==> Lactate + NAD+


where reaction (1) is catalyzed by your ATPase, reaction (2) by pyruvate kinase, and reaction (3) by lactate dehydrogenase. Loss of NADH (as monitored by absorbance at 340 nm) is quantifiably proportional to ATP hydrolysis rates because this set of reactions begins with equilibria strongly favoring the forward direction and neither reactions (2) or (3) are rate-determining.


Calculating the ATPase Rate.


Total ATPase rate = (rate of A340 signal loss)/(pathlength*6.23mM^-1cm^-1)


By applying the above formula, you'll get some rate expressed in mM per unit time. For the molar activity (i.e., rate per mole of enzyme) simply divide this rate by the enzyme concentration you used in the assay. This will give you a hydrolysis rate of per unit time per enzyme.


Procedure

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