Enzymic Studies on the Biosynthesis of Serotonin in Mammalian Brain

Abstract

Abstract A rapid, convenient and sensitive assay method for measuring tryptophan 5-monooxygenase in mammalian brain is developed with l-tryptophan-carboxyl-14C as substrate. The method is based on the differing affinity of aromatic l-amino acid decarboxylase toward l-tryptophan and 5-hydroxy-l-tryptophan (Km values for l-tryptophan and 5-hydroxy-l-tryptophan are approximately 1.4 x 10-2 m and 5.4 x 10-6 m, respectively). The reaction is carried out in the presence of an excess amount of the decarboxylase, and the radioactive CO2 preferentially liberated from 5-hydroxy-l-tryptophan, which is formed from l-tryptophan-14C by the action of tryptophan 5-monooxygenase, is measured. Tryptophan 5-monooxygenase activity is detected in both the soluble and particulate fractions from the guinea pig brain stem. When relatively low concentrations (below 10-5 m) of l-tryptophan-(side chain-1,2,3-14C) are used as substrate and incubated with the supernatant or the crude mitochondrial fractions in the presence of monoamine oxidase inhibitor, the radioactive CO2 evolved is almost entirely accounted for by the formation of serotonin-14C. Only small amounts of tryptamine-14C are produced and very little 5-hydroxy-l-tryptophan-14C accumulates in the reaction mixture. Properties of tryptophan 5-monooxygenase in both the supernatant and crude mitochondrial fractions are examined and the appropriate conditions for the assay are described.