ATPase (Na+/K+) Pig Heart Enzymatic LeadHunter Assay – TW

Item: 107710

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OVERVIEW

DOCUMENTS/REFERENCES

Biological Information

Background Information:

Na+/K+-ATPase has been found in the plasma membrane of nearly all eukaryotic cells, although its density per cell type differs, e.g., low in red blood cells, medium in cardiac ventricular muscle and high in mammalian kidney and brain tissues. It is the enzymatic machinery in the Na+/K+ pump which uses the energy from ATP hydrolysis to transport Na+ and K+ across the cell membranes. The pump plays an important role in the regulation of normal cell homeostasis, cell differentiation and cell proliferation. It is known that inhibition of Na+/K+-ATPase activity by cardiac glycosides is the basis for treating atrial fibrillation; this enzyme appears to represent the digitalis receptor.

Family:

Transporter

Class:

ATPase

Organism:

Pig

Construct Details:

Endogenous

Assay Information

Assay Type:

Biochemical

Assay Sub Type:

Enzymatic

Functional Mode:

Antagonist

Detection Method:

Spectrophotometry

Measured Response:

Absorbance

Testing Information

Procedure Summary:

Na+/K+-ATPase prepared from pig heart is used. Test compound and/or vehicle is preincubated with 8 µg/ml enzyme‡ in modified Tris-HCl buffer pH 7.0 for 15 minutes at 37°C. The reaction is initiated by addition of 100 μM ATP for another 60 minute incubation period. An aliquot is removed for determination of the amount of inorganic phosphate formed spectrophotometrically at 630 nm by biomol green reagent. Compounds are screened at 10 μM.

Substrate:

100 µM ATP

Incubation:

60 min at 37°C

Control Inhibitor:

Ouabain

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

Turnaround Time

Standard:

15 Business Days

Reference Compound Data

PubChem ID

439501

Name

Ouabain

Measurement

2μM - IC50

PubChem ID

34174823

Name

Bufalin

Measurement

0.11μM - IC50

PubChem ID

Name

p-Chloromercuribenzene-sulfonic acid (p-CMBS)

Measurement

0.78μM - IC50

Additional Information

Brand:

LeadHunter

Testing Location:

Taiwan - Taipei