Sauer:ATPase assays

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(1)  ATP <==> ADP + Pi
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(1)  ATP &hArr; ADP + P<sub>''i''</sub>
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(2)  ADP + phosphoenolpyruvate <==> ATP + pyruvate
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(2)  ADP + phosphoenolpyruvate &hArr; ATP + pyruvate
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(3)  pyruvate + NADH + H+ <==> lactate + NAD+
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(3)  pyruvate + NADH + H<sup>+</sup> &hArr; lactate + NAD<sup>+</sup>
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Total ATPase rate = (rate of A340 signal loss)/(pathlength*6.23mM^-1cm^-1)
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<math>T\!otal\mbox{ }AT\!P\!ase\mbox{ }rate = \frac {rate\mbox{ }of\mbox{ }A_{340nm}\mbox{ }signal\mbox{ }loss} {pathlength * 6.23 mM^{-1}cm^{-1}}</math>
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'''Procedure.''' In our lab, we use a 96-well plate reader that measures A340 over time in a 50uL reaction volume.   
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'''Procedure.''' In our lab, we use a 96-well plate reader that measures A<sub>340 nm</sub> over time in a 50 &mu;L reaction volume.   
''will get back to this...''
''will get back to this...''
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<math><math>Insert formula here</math></math>
 

Revision as of 16:03, 10 November 2006

SAUER LAB

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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 referenced in Norby, JG (1988) Meth enzy, 156, 116-9. The basic principle can be summarized below:


(1) ATP ⇔ ADP + Pi

(2) ADP + phosphoenolpyruvate ⇔ 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.


T\!otal\mbox{ }AT\!P\!ase\mbox{ }rate = \frac {rate\mbox{ }of\mbox{ }A_{340nm}\mbox{ }signal\mbox{ }loss} {pathlength * 6.23 mM^{-1}cm^{-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 in units of per unit time per enzyme.


Procedure. In our lab, we use a 96-well plate reader that measures A340 nm over time in a 50 μL reaction volume.


will get back to this...

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