Tribioscience Catechol O-Methyl Transferase, Enzyme Activity (TBP0061)

Tribioscience Catechol O-Methyl Transferase, Enzyme Activity (TBP0061)

Research Use Only. Not intended for human use.

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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%

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. 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).
2. 10 mM Magnesium chloride, in distilled water (0.952 mg/ml). 100µl.
3. 5 mM Dithiothreitol (0.61 mg/ml) in distilled water. 100 µl.
4. 0.5 M Tris-HCl buffer, pH 8.0 in distilled water. 200 µl.
5. 10-50 mM 3,4 Dihydroxybenzoic acid, (1.56-7.81 mg/ml) in water. 200 µl.
6. 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.

7. 1% Bovine serum albumin (BSA) solution. Dissolve 1.0 g BSA in 100 ml distilled water. Albumin should be of highest purity.
8. COMT (enzyme) solution: Prepare a suitable dilution of the enzyme using cold 1% BSA. Prepare fresh prior to assay.
9. Hydrochloric acid (conc.).
10. Toluene: 3-methyl butanol, 7:3 (v/v).

Procedure

1. Pipette 100 µl of the assay solution in the assay tube.
2. Add 100 µl of a suitable dilution of COMT (enzyme) to the assay tube at zero time. Mix.
3. Incubate the assay tube for 10 min at 37°C.
4. Terminate the reaction by adding 0.1 ml HCl to the assay tube.
5. Extract radioactive products into 6 ml of a mixture of toluene: 3 methylbutanol (7:3 v/v) by shaking for 20 seconds.
6. 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.