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BioAssay: AID 686936

In vitro LuxO ATPase Assay Measured in Biochemical System Using Plate Reader - 2132-11_Inhibitor_Dose_DryPowder_Activity

A modified coupled-enzyme assay measured the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxOD47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. ATP hydrolysis rates more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 686936
Data Source: Broad Institute (2132-11_Inhibitor_Dose_DryPowder_Activity)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2013-04-13
Hold-until Date: 2013-07-17
Modify Date: 2013-07-17

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2
Depositor Specified Assays
AIDNameTypeComment
588521Broad Institute Quorum Sensing in Vibrio cholera: CqsS/LuxQ Agonist Probe ProjectsummarySummary
686945Broad Institute Quorum Sensing in Vibrio cholera: LuxO Inhibitor Probe Projectsummary
Description:
Keywords: LuxO, cholera, ATPase, kinase

Assay Overview:

A modified coupled-enzyme assay measured the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxOD47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. ATP hydrolysis rates were inferred from the absorbance change observed (aNADH,340 -a-NAD,340 = 6220 M-1 cm-1 for NADH). The rates of ATP hydrolysis by LuxOD47E were measured in reactions containing 100 mM sodium phosphate buffer pH 7.4, 5 mM MgCl2, 0.2 mM NADH, 1 mM PEP, 5-20 units of PK/LDH mix (Sigma), and 10 mM LuxOD47E. ATP and inhibitors were added to the reactions at indicated concentrations. The rate of ATP hydrolysis was monitored for 5 minutes. Data were fitted using GraphPad Prism to obtain the kinetic parameters. Percent ATPase inhibition was calculated.


Expected Outcome: A compound that inhibits ATPase activity of LuxO will lead to a decrease in ATP hydrolysis, measured as a lower OD value.
Protocol
A modified coupled-enzyme assay was used to measure the rate of ATP hydrolysis by LuxOD47E. Briefly, ADP released from ATP by LuxO D47E is reacted with phosphoenolpyruvate (PEP) to form pyruvate using pyruvate kinase (PK). Pyruvate is reacted with NADH to form NAD and lactate using lactate dehydrogenase (LDH). The rate of NAD production is followed at 340 nm using a spectrophotometer. The rates of ATP hydrolysis by LuxOD47E were measured in reactions containing 100 mM sodium phosphate buffer pH 7.4, 5 mM MgCl2, 0.2 mM NADH, 1 mM PEP, 5-20 units of PK/LDH mix (Sigma), and 10 microM LuxOD47E. ATP was added at a final concentration of 2.5 mM. The rate of ATP hydrolysis was monitored for 5 minutes. The values reported are delta OD340/min.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AVG_DELTA_OD_AT_100um (100μM**)Float
2AVG_DELTA_OD_AT_25um (25μM**)Float
3AVG_DELTA_OD_AT_1um (1μM**)Float
4DELTA_OD_AT_100um_TEST1 (100μM**)Float
5DELTA_OD_AT_25um_TEST1 (25μM**)Float
6DELTA_OD_AT_1um_TEST1 (1μM**)Float
7DELTA_OD_AT_100um_TEST2 (100μM**)Float
8DELTA_OD_AT_25um_TEST2 (25μM**)Float
9DELTA_OD_AT_1um_TEST2 (1μM**)Float
10DELTA_OD_AT_100um_TEST3 (100μM**)Float
11DELTA_OD_AT_25um_TEST3 (25μM**)Float
12DELTA_OD_AT_1um_TEST3 (1μM**)Float
13DELTA_OD_AT_100um_TEST4 (100μM**)Float

** Test Concentration.
Additional Information
Grant Number: R03 MH094166-01

Data Table (Concise)
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