Catechol O-Methyl Transferase, Enzyme Activity (TBP0061)
$420.00 – $1,680.00
SKU | Stock | SIZE (mg) | Price | Quantity | ||
---|---|---|---|---|---|---|
TBP0061-1MG | Yes | 1 MG | $420.00 | |||
TBP0061-5MG | Yes | 5 MG | $1,680.00 |
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Description
Product Details
Form: Freeze-dried powder
Solubility: Distilled water or dilute buffer
Stability: Store at -20° C (-4° F)
Activity: 4000 U/mg protein
Protein: 90%
Contaminants: PNMT <0.01%
Catalog No.: 064A4000
Unit Definition
The amount of enzyme which will catalyze the methylation of one nanomole of dihydroxybenzoic acid per hour at 37°C, pH 8.0, using S-adenosyl-L-(methyl 14C)-methionine as the methyl donor.
Assay Method
Enzyme activity is determined by measuring the amount of 14C incorporated into the methylated substrate (3,4- Dihydroxybenzoic acid).
Applications
The enzyme, catechol O-methyltransferase (COMT) (EC 2.1.1.6) can O-methylate a wide variety of substituted catechols such as dopamine, epinephrine and norepinephine. This is an important step in the metabolic detoxification of catecholamines. A representative reaction is shown below:
Reagents
- 1 mM S-Adenosylmethionine (SAM), (0.435 mg/ml) 100µl. (This product must be of highest purity and must not contain traces of S-adenosylhomocysteine).
- 10 mM Magnesium chloride, in distilled water (0.952 mg/ml). 100µl.
- 5 mM Dithiothreitol (0.61 mg/ml) in distilled water. 100 µl.
- 0.5 M Tris-HCl buffer, pH 8.0 in distilled water. 200 µl.
- 10-50 mM 3,4 Dihydroxybenzoic acid, (1.56-7.81 mg/ml) in water. 200 µl.
- 14C S-Adenosylmethionine, (55mCi/mMole). Distilled water is added to this solution to make 1 ml. It must be kept on ice until needed. Use 10µCi in the assay mixture.
Note: The assay solution used for COMT assay is prepared by mixing the above reagents in the amounts indicated.
- 1% Bovine serum albumin (BSA) solution. Dissolve 1.0 g BSA in 100 ml distilled water. Albumin should be of highest purity.
- COMT (enzyme) solution: Prepare a suitable dilution of the enzyme using cold 1% BSA. Prepare fresh prior to assay.
- Hydrochloric acid (conc.).
- Toluene: 3-methyl butanol, 7:3 (v/v).
Procedure
- Pipette 100 µl of the assay solution in the assay tube.
- Add 100 µl of a suitable dilution of COMT (enzyme) to the assay tube at zero time. Mix.
- Incubate the assay tube for 10 min at 37°C.
- Terminate the reaction by adding 0.1 ml HCl to the assay tube.
- Extract radioactive products into 6 ml of a mixture of toluene: 3 methylbutanol (7:3 v/v) by shaking for 20 seconds.
- The aqueous phase is allowed to settle and then discarded, while the upper organic phase is saved and dried and then taken up in 1 ml ethanol and 14C radioactivity is counted in a scintillation spectrometer.
Additional information
SIZE (mg) | 1 MG, 5 MG |
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