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DNA Modifying Enzymes and Cloning >
Phosphatases and Sulfurylases >
Apyrase |
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
Apyrase is an ATP diphosphohydrolase. It catalyses the removal of the gamma phosphate from ATP and the beta phosphate from ADP. The phosphate from AMP is not removed.
10 units = 10,000 milliunits
Source:
This preparation is purified from K. lactis containing a clone of the potato apyrase gene (1).
Reagents Supplied:
Succinate Buffer for control purposes (10X)
Reaction & Storage Conditions
Unit Definition:
One unit is defined as the amount of enzyme that catalyzes the conversion of 1 µmol of ATP to ADP in one minute at 30°C in a total reaction volume of 40 µl.
Unit Assay Conditions:
1X Succinate Buffer, 40 mM sodium succinate (pH 6.5), 4 mM CaCl2 and 1 mM ATP at 30°C.
Concentration:
50,000 milliunits/ml
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
0.1 mM CaCl2
0.1 mM DTT
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- The activity of Apyrase at pH 7.5 in a Tris Buffer is approximately 80% of the activity at pH 6.5. Magnesium can substitute for calcium in the reaction. The ratio of ATPase:ADPase is 4:1 with this preparation of Apyrase.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Phosphatase Contamination:
After incubation of 1 unit of Apyrase with 0.05 µmol p-nitrophenol phosphate for 20 hours at 37°C, no phosphatase activity could be detected by spectrophotometric analysis.
Nuclease Contamination:
Incubation of 1 unit of Apyrase for 6 hours in the recommended assay buffer with Lambda-HindIII Digest and 100 bp DNA Ladder revealed no detectable endonuclease activity as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of 50 µl reaction containing 1 unit of Apyrase with 1 µg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/µg) for 6 hours at 37°C released < 0.5% of the total radioactivity.
References
- Ganatra, M., Hough, D. and Taron, C.H., unpublished observations.
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