Effects of norepinephrine on pineal tryptophan hydroxylase, using an improved [14C]CO2-trapping microtechnique

The synthesis of many biogenic amines involves hydroxylation and/or decarboxylation reactions. These enzymes are often measured by trapping and be determining the concentrations of their end products [14C]CO2, from carboxyl-labeled substrates. An improved [14C]CO2-trapping technique has been developed for microassay of tryptophan hydroxylase activity. This technique allows hydroxylation and decarboxylation reactions to be carried out sequentially in microculture tubes containing reaction mixtures in less than 50 microliter. The incubation vessel consists of two microculture tubes connected by a latex tubing serving as a sleeve. One of the two microculture tubes were used to carry out enzymatic reactions and the other one was filled with CO2-trapping agent to trap [14C]CO2. The utility of this technique was tested by measuring the effects of biogenic amine on tryptophan hydroxylase in bovine pineal explants in culture. The bovine pineal tryptophan hydroxylase was found to have a specific activity of 8.93 pmol/mg protein/min, and its activity was linear with respect to both incubation time and to protein concentrations. In addition, treatment with norepinephrine of both rat pineal gland and bovine pineal explants in culture for 2, 4, and 6 hr had no effect on the activity of tryptophan hydroxylase. It is believed that this technique may be adapted easily to measure numerous CO2-generating enzymatic reactions whenever the amounts of protein or the size of tissue demands the application of a microtechnique.