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Phenylethanolamine N-Methyl Transferase (PNMT)

(S-adenosyl-L-methionine:phenylethanolamine-N-methyltransferase; EC 2.1.1.28)

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme which catalyzes the N-methylation of norepinephrine thereby resulting in the formation of epinephrine as shown below:

                                               PNMT
Norepinephrine + S-Adenosyl methionine (SAM) -------> Epinephrine 
The mechanism involves transfer of an active methyl group from S-adenosylmethionine (SAM) to the primary amino group of norepinephrine. Although it is primarily localized in the adrenal medulla, PNMT activity has also been demonstrated in the brain and heart tissues of several mammalian species including humans (Connett, R.J. and Kirshner, N., J. Biol. Chem., 245, 329, 1970; Eagles, P.A.M. and Iqbal, M, Brain Research, 80, 177, 1974). PNMT purified from ox, rat and rabbit adrenal medulla have molecular weights in the range of 37,000-38,000. Analysis of PNMT activity could provide valuable information in the evaluation of catecholamine metabolism.


1.Source: Bovine Adrenal Medulla
   Form: Freeze-dried 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20 C; -4 F 
   Activity: 50-100 U/mg protein 
   Catalog No.: 103A0050 
$20.00/mg


The amount of enzyme which will convert one nanomole of normetanephrine to metanephrine per hour at pH 8.5 at 37°C.


 Enzyme activity is determined by measuring the amount of 14C
 incorporated into the metanephrine formed during the
 reaction. S- adenosyl-L-(methyl 14C)-methionine serves as a
 methyl donor.

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  1. 1.0 mM S-adenosylmethionine (SAM), (0.435 mg/ml). 20 µl. (This product must be of highest purity and must not contain traces of S-adenosylhomocysteine).
  2. 1.0 M Tris-HCl, pH 8.5 in distilled water. 200 µl.
  3. 30 mM D,L Normetanephrine, (5.1 mg/ml) in distilled water. 100 µl.
  4. 14C S-adenosylmethionine, 10 µCi (55 mCi/mM). Distilled water is added to this solution to make 0.5 ml. This solution must be kept on ice until use. Note: The assay solution used for PNMT assay is prepared by mixing the above four reagents in the amounts indicated.
  5. 1% Bovine serum albumin (BSA) solution.
  6. 0.5 M Sodium borate, (100.65 g/L), pH 10.0.
  7. Toluene:isoamyl alcohol (3:2, v/v).
  8. PNMT (enzyme) solution. Prepare a suitable dilution of the enzyme using cold 1% BSA. Prepare fresh prior to assay.



  1. Pipette 50 µl of the assay solution in the assay tube.
  2. Add 50 µl of a suitable dilution of PNMT (enzyme) to the assay tube at zero time. Mix well.
  3. Incubate the assay tube for 10 minutes at 37¡C.
  4. Terminate the reaction by adding 1 ml 0.5 M Sodium borate to the assay tube.
  5. Extract the radioactive products into 6 ml of a mixture of toluene:isoamyl alcohol (3:2 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.



Note: 6=Conversion Factor from 10 minutes incubation to 60 minutes (see Unit Definition).
 

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