Steady State ATPase Assays Coupled Enzyme System

The coupled enzyme ATPase assay is based on the conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase (PK) coupled to the conversion of pyruvate to lactate by lactate dehydrogenase (LDH). The latter step requires NADH which is oxidized to NAD+. NADH absorbs strongly at 340 nm but NAD+ does not, enabling the ultilization of NADH to be followed by monitoring absorbance at 340 nm. The decrease in OD340 can be converted into ATPase activity where 1 molecule of NADH oxidized to NAD+ corresponds to the production of 1 molecule of ADP by the motor ATPase. (From Kinesin site, Liz Greene and Steve Henikoff et al).

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Last update 20-Jan-2005, Rating n/a of 0 votes.