The amount of enzyme which will convert one nanomole of histamine to methyl histamine per hour at pH 8.5 at 37°C.
Applications
(HNMT) (EC 2.1.1.8) is the enzyme which catalyzes the n-methylation of histamine as follows:
The mechanism involves the transfer of an active methyl group from S-Adenosyl methionine (SAM) to histamine. Histamine is present in most of mammalian tissues and HNMT is the enzyme responsible for inactivation of histamine in mammals. Methylation is major route of histamine metabolism. HNMT has been used to measure histamine by radio-enzymatic method. HNMT has been purified from rat kidney. Molecular weight equals 33,400, pH optimum is 8.00-8.25. We have also purified it from bovine kidney which seems to be very similar to rat kidney.
Reagents
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.)
0.125 M Potassium Phosphate, pH 7.8, in distilled water. 200 µl.
0.01 M Histamine in distilled water. 100 µl.
14C S-adenosylmethionine, 10 µCi (55 mCi/mMole). 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 HNMT assay is prepared by mixing the above four reagents in the amounts indicated.
1% Bovine serum albumin (BSA) solution.
0.5 M Sodium borate, (100.65 g/L), pH 10.0.
Toluene:isoamyl alcohol (3:2, v/v).
HNMT (enzyme) solution. Prepare a suitable dilution of the enzyme using cold 1% BSA. Prepare fresh prior to assay.
Procedure
Pipet 50 µl of the assay solution in the assay tube.
Add 50 µl of a suitable dilution of HNMT (enzyme) to the assay tube at zero time. Mix well.
Incubate the assay tube for 10 minutes at 37°C.
Terminate the reaction by adding 1 ml of 0.5 M Sodium borate to the assay tube.
Extract the radioactive products into 6 ml of a mixture of toluene:isoamyl alcohol (3:2, 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.
Note: The assay should be carried out in borosilicate cultured tubes, since Histamine and methyl Histamine are absorbed by glass surfaces.
