| Assay Method Information | |
| | Assaying Cardiac Muscle Myosin II |
| Description: | The cardiac muscle myosin II actin-activated ATPase assay is a biochemical assay. Specifically, it is an NADH (nicotinamide adenine dinucleotide)-coupled ATPase assay that relies on NADH fluorescence as a readout. Cardiac myosin is a mechanochemical energy transducer that hydrolyzes ATP to generate force in the presence of its activator, F-actin. The resulting ADP is regenerated to ATP by pyruvate kinase (PK) that transforms one molecule of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH) that, in turn, oxidizes one molecule of NADH to NAD. Therefore, the decrease in NADH concentration as a function of time equals the ATP hydrolysis rate. Bovine cardiac myosin is obtained from a commercial source, Cytoskeleton. PK, LDH, ATP, PEP, and NADH are obtained from Sigma. F-actin is prepared in house from Rabbit Muscle Acetone Powder. The assay is run at 25° C. in 384 well black-wall polystyrene microplates with a total volume of 20 μl per well. NADH fluorescence is monitored for 30 minutes with an EnVision Multimode Plate Reader. The slope of the fluorescence response, which is proportional to the reaction rate, is determined by simple linear regression. Final assay conditions are 300 nM cardiac myosin, 10 μM actin, 40 U/ml LDH, 200 U/ml PK, 220 μM NADH, 1 mM PEP, 1 mM ATP in a buffer containing 10 mM 3-(N-morpholino)propanesulfonic acid (pH=7.0), 2 mM MgCl2, 0.15 mM ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.1 mg/mL bovine serum albumin, 0.5% (V/V) dimethyl sulfoxide (DMSO) and 1 mM dithiothreitol. Prior to testing the inhibitory activity of the compounds, a two-fold dilution series starting at 10 mM compound concentration is prepared in DMSO. Subsequently, 100 nl is transferred to each well of the measuring plate containing a mixture of myosin, LDH and PK. The enzymatic reaction is started with the addition of a mixture containing ATP, PEP, NADH and actin. The highest final compound concentration is 50 uM. 20 μM para-aminoblebbistatin in 0.5% DMSO serves as the positive control and 0.5% DMSO alone is the negative control. Reaction rates are determined by using the fluorescence responses of a dilution series of NADH included in all plates and plotted as a function of inhibitor concentration. All measurements are carried out in triplicate. Inhibitory constants are determined by fitting the 16 point dose-response data to a quadratic equation corresponding to a simple one-to-one binding model. Small molecules showing no observable inhibition at or below their solubility are reported as inactive. |
| Affinity data for this assay | |
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