Dopamine (DA) was measured in various tissue extracts as [3H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [3H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.
A method for determination of catecholamines using the enzyme catechol-O-methyl transferase (COMT, EC 2.1.1. I)* and radiolabeled S-adenosylmethionine (SAMe) was described originally by Axelrod and Tomchick (1). COMT forms methoxy derivatives with all catecholamines, so that in order to distinguish between dopamine (DA) and norepinephrine (NE), special separation procedures were devised. These include oxidation of the P-hydroxy side chain by periodate followed by selective solvent extraction, alumina chromatography, treatment with acetic anhydride, tic, etc. Some of the more recent reports describe these procedures (2-14).
The procedure to be described involves two enzyme systems: (i) COMT and S-' To whom correspondence should be addressed.