Tryptophan hydroxylase [tryptophan 5-monooxygenase, L-t top an,tetrahydropterin:oxygen oxidoreductase (5-hydroxylating), EC 1.14. 16.4] is activated by phosphorylating conditions (ATP-Mg2+) in a calcium-dependent, cyclic AMP-independent manner. Addition to the hosphorylation reaction of certain antipsychotic drugs thattind to calmodulin, the heat-stable calcium-binding protein, prevents the activation of tryptophan hydroxylase by ATP-Mg+ in a concentration-dependent fashion. Externa addition of purified calmodulin protects the enzyme from the drug-induced effects.Calmodulin-free tryptophan hydroxylase prepared by affinity chromatography on fluphetrazine-Sepharose is not activated by ATP-Mg2+ whereas addition of calmodulin to calmodulin-free enzyme restores the responsiveness of the hydroxylase to ATP-Mg2+ only in the presence of Ca2+. These results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.Tryptophan hydroxylase [tryptophan 5-monooxygenase, Ltryptophan,tetrahydropterin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (1). Recent studies in our laboratory (2) and in the laboratories of others (3, 4) have demonstrated that this important enzyme can be activated by ATP-Mg2+. This apparent phosphorylation of tryptophan hydroxylase is cyclic nucleotide independent and calcium dependent (2, 3). Implicit in these studies was a potential role for a protein kinase that was dependent on calmodulin, the heat-stable calcium-binding regulator protein (5 Tryptophan hydroxylase was assayed by a modification (7) of the method of Friedman et al. (8). The standard reaction mixture contained the following substituents in a total volume of 50 ,ul: 120-130 ,ug of tissue extract, 50mM Tris-HCl (pH 7.4), 2.0 mM dithiothreitol, 100 ,uM Ca2+, 400 international units of catalase, 0.1 mM 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin (subsaturating), and 0.4 mM L-tryptophan (saturating). Reactions were started by addition of all substituents all at once to the enzyme. Each reaction component was prepared in 50 mM Tris-HCl (pH 7.4), except for the 6-methyltetrahydropterin, which was dissolved in 10 mM HCO and catalytically reduced under hydrogen in the presence of platinum oxide as described (9). Each tube was incubated for 15 min at 37°C, and the reactions were terminated by addition of 10,l of 6 M perchloric acid. Precipitated protein was removed by centrifugation, and a 40-,ul aliquot of the supernatant fraction was added to 100 ,ul of 8 M HCL. The fluorescence of the solution was measured in an Aminco-Bowman spectrophotofluorometer at excitation-emission wavelengths of 295-540 nm. The amount of 5-hydroxytryptophan formed enzymatically was calculated from a standard curve of authentic 5-hydroxytryptophan carried through the entire procedure. The hydroxylation of tryptophan is linear with respect to the time parameters and protein concentra...
