A sensitive enzymatic assay for dopamine- -hydroxylase.

Abstract

A sensitive and convenient procedure for the determination of dopamine-β-hydroxylase (DBH) activity in homogenates of sympathetically innervated organs is described. The assay uses either phenylethylamine or tyramine as substrate and is based on the sequential conversion of the product of the DBH reaction to a radioactively labeled N-methyl derivative by reaction with partially purified bovine adrenal phenylethanolamine-N-methyltransferase in the presence of S-adenosyl-l-methionine methyl- 14 C. The N-methyl derivatives are separated by solvent extraction and their radioactivity is determined. The identity of the reaction products has been determined chromatographically, and the characteristics of the two enzymatic steps in the assay procedure are defined. Endogenous tissue inhibitors of DBH must be inactivated by cupric ions prior to determination of the enzyme activity. DBE activity in sympathetically innervated tissues of the rat has been measured.