Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain.

Abstract

Tryptophan hydroxylase [EC 1.14.16.4; Ltryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from the region of the raphe nucleus of rat midbrain by sequential column chromatography and disc-gel electrophoresis. In electrophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to electrophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chlorophenylalanine. Antibodies produced to each of the three bands crossreacted by immuno double diffusion and electrophoresis with each other and homogenates of raphe nuclei; they completely inhibited enzyme activity only of tryptophan hydroxylase. Tryptophan hydroxylase was localized by light and electron immunohistochemistry to serotonin neurons of the raphe. Ultrastructurally, in cell bodies, the enzyme was distributed in cytoplasm and in association with endoplasmic reticulum and Golgi apparatus. In dendrites and axons, it was associated with microtubules. Tryptophan hydroxylase in brain is only neuronal and cytoplasmic, exists in multiple forms, and is associated with microtubules, suggesting it may be transported from sites of synthesis in cell body into axons.The enzyme, tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropterine:oxygen oxidoreductase (5-hydroxylating)] catalyzes the initial and probably rate-limiting step in the biosynthesis of the neurotransmitter serotonin (5HT) (1, 2). In brain, the enzyme has been presumed to be contained only within those specific neurons which synthesize, store, and release 5HT (3). The evidence is indirect and based on the reasonable assumption that those neurons in which 5HT can be visualized by their specific histofluorescence (4, 5) should contain the enzyme. Consistent with the surmise are biochemical data demonstrating the presence of high levels of tryptophan hydroxylase activity in brain regions containing the cell bodies of 5HT neurons (3) or richly innervated by their processes (3). It has also been proposed, on indirect evidence, that tryptophan hydroxylase exists in different forms, differing either in susceptibility to inhibition by drugs (6-8) or possibly, in their relationship to subcellular organelles (3).Direct information of the biochemical structure and regional and subcellular localization of the enzyme has been restricted by limited success (9, 10) in obtaining purified tryptophan hydroxylase suitable for the development of specific antibodies for immunohistochemistry. Recently the development in our laboratory of techniques for the purification and immunohistochemical localization of tyrosine hyAbbreviation: 5HT, serotonin.

5-HT2A receptor antagonist M100907 reduces serotonin synthesis: An autoradiographic study

“…The action through 5-HT 2A receptors is probably also related to 5-HT dopamine interaction because blocade of 5-HT 2A receptors on dopaminergic neurons could result in higher release of dopamine which could influence 5-HT synthesis [35]. The tryptophan hydroxylase (TPH) is "mostly" expressed in the cell bodies found in raphe nuclei (e.g., [36]), but there is also substantial evidence that TPH is present in the serotonergic terminals [62,18,59], and that the synthesis of 5-HT is occurring in those terminals (e.g., [49,33,11,51]). …”

Characterization of a Stable Form of Tryptophan Hydroxylase from the Human Parasite Schistosoma mansoni

“…The three enzymes catalyze similar hydroxylation reactions and share a distinctive requirement for tetrahydrobiopterin (BH 4 ) and non-heme ferrous iron as cofactors (2). Studies of TPH have been hampered by the extreme instability of the enzyme (5)(6)(7)(8)(9)(10)(11) and the difficulty in obtaining high levels of purified active enzyme from either native or heterologous sources (10 -17). Sequence analyses of TPH cDNAs derived from mammalian and other vertebrate species (13, 18 -23) revealed that TPH shares high overall sequence homology with the other two members of the hydroxylase family (1,2).…”

Serotonergic neurons in the peripheral nervous system: identification in gut by immunohistochemical localization of tryptophan hydroxylase.

“…After 3 weeks' growth, cultures were fixed in picric acid/formalin and processed as whole mounts. The sections of rat intestine, the whole mounts of longitudinal muscle with attached'myenteric plexus from guinea pigs, and the cultures from mice were incubated with rabbit antiserum to tryptophan hydroxylase (purified from raphe nuclei of rat brain), goat antiserum to rabbit IgG, and peroxidase-antiperoxidase complex (18,20). Peroxidase activity was then localized with 3,3'-diaminobenzidine and hydrogen peroxide (18,20).…”

“…The sections of rat intestine, the whole mounts of longitudinal muscle with attached'myenteric plexus from guinea pigs, and the cultures from mice were incubated with rabbit antiserum to tryptophan hydroxylase (purified from raphe nuclei of rat brain), goat antiserum to rabbit IgG, and peroxidase-antiperoxidase complex (18,20). Peroxidase activity was then localized with 3,3'-diaminobenzidine and hydrogen peroxide (18,20). In this bridge technique, the unlabeled rabbit antibody to tryptophan hydroxylase combines with the enzyme in the tissue, the goat antiserum bridges the rabbit antibody to the peroxidase-antiperoxidase complex, and the peroxidase activity localizes the tryptophan hydroxylase.…”