Decarboxylation of L-3,4-dihydroxyphenylalanine to dopamine, and of 5-hydroxytryptophan to serotonin, is catalyzed by aromatic L-amino acid decarboxylase. A single enzyme may be responsible for both activities. This assay permits simultaneous determination of both activities.
The substrates and products just noted were separated on an Ultrasphere I.P. Cig column (4.6 mm X 250 mm, 5 /¿m). The mobile phase contained 75 mM sodium phosphate (pH 2.75), 1 mM sodium octylsulfate, 500 fiM EDTA, and 13% (v/v) acetonitrile. Quantitation was by electrochemical detection of the products using 0.75 V versus an Ag/AgCl reference electrode.
The standard incubation mixture contained 0.2 M phosphate buffer (pH 7.5), 0.02 m M pyridoxal phosphate, 0.1 m M pargyline, 0.2 m M L-dihydroxyphe-nylalanine, 0.1 mM 5-hydroxytryptophan, and enzyme in a total volume of 200 fiL. After incubation at 37°C for 30 minutes, the reactions were terminated by addition of 800 /xL of chilled 0.1 M perchloric acid containing 0.1 mM sodium metabisulfite and 0.2 mM EDTA. After centrifugation, 10 /xL aliquots were used for HPLC analysis.
Tissue homogenates in 5 volumes of chilled 0.05 M phosphate buffer (pH 7.4) were prepared from the liver, kidney, adrenal, brain, heart, lung, and small and large intestine of rats.
Was this article helpful?