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Brain Tryptophan Hydroxylase

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Brain Tryptophan Hydroxylase Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3575-3579, September 1975 Cell Biology Brain tryptophan hydroxylase: Purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain (immunocytochemistry/microtubules/raphe nucleus/serotonin/tryptophan 5-monooxygenase) TONG HYUB JOH, TADAHIRO SHIKIMI, VIRGINIA M. PICKEL, AND DONALD J. REIS Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, 1300 York Avenue, New York, N.Y. 10021 Communicated by Seymour S. Kety, June 19, 1975 ABSTRACT Tryptophan hydroxylase [EC 1.14.16.4; L- droxylase (11-15), a closely related enzyme which subserves tryptophan, tetrahydropteridine:oxygen oxidoreductase (5- the biosynthesis of catecholamine neurotransmitters, has en- hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from couraged us in this study to attempt purification and local- the region of the raphe nucleus of rat midbrain by sequential ization of tryptophan hydroxylase in brain. column chromatography and disc-gel electrophoresis. In elec- trophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to elec- MATERIALS AND METHODS trophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chloro- phenylalanine. Antibodies produced to each of the three Materials. L-Tryptophan, dithiothreitol, catalase, and bands crossreacted by immuno double diffusion and electro- pyridoxal-5'-phosphate were purchased from Sigma Chemi- phoresis with each other and homogenates of raphe nuclei; cal Co.; 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahy- they completely inhibited enzyme activity only of trypto- dropteridin from Aldrich Chemical Co.; NCS-Tissue Solubil- phan hydroxylase. Tryptophan hydroxylase was localized by izer from Amersham-Searle Corp.; L-[1-'4C]tryptophan light and electron immunohistochemistry to serotonin neu- from New England Nuclear Corp. Sepharose 4B and Sepha- rons of the raphe. Ultrastructurally, in cell bodies, the en- zyme was distributed in cytoplasm and in association with dex G-200 were obtained from Pharmacia Fine Chemical, endoplasmic reticulum and Golgi apparatus. In dendrites Inc., and acrylamide H.P. and N,N'-methylene bisacrylam- and axons, it was associated with microtubules. Tryptophan ide H.P. from Canalco, Inc. hydroxylase in brain is only neuronal and cytoplasmic, exists Tryptophan hydroxylase was purified from the region of in multiple forms, and is associated with microtubules, the raphe nuclei of the midbrain of 260 male Sprague- suggesting it may be transported from sites of synthesis in Dawley rats dissected according to the procedure of Degu- cell body into axons. chi et al. (16). Aromatic L-amino-acid decarboxylase (EC The enzyme, tryptophan hydroxylase [EC 1.14.16.4; L-tryp- 4.1.1.28; aromatic-L-amino-acid carboxy-lyase) was partially tophan, tetrahydropterine:oxygen oxidoreductase (5-hydrox- purified from hog kidney of DEAE-Sephadex column chro- ylating)] catalyzes the initial and probably rate-limiting step matography by the method of Christenson et al. (17). in the biosynthesis of the neurotransmitter serotonin (5HT) Enzyme Assay. Tryptophan hydroxylase activity was as- (1, 2). In brain, the enzyme has been presumed to be con- sayed by a modification of the method of Ichiyama et al. tained only within those specific neurons which synthesize, (10), in which the rate of conversion of L-[1-'4C]tryptophan store, and release 5HT (3). The evidence is indirect and to '4CO2 was measured. The assay mixture contained 200 Al based on the reasonable assumption that those neurons in of enzyme preparation, 50 ,l of 1 M Tris-acetate buffer, pH which 5HT can be visualized by their specific histofluores- 7.6, 10 Al of 30 mM dimethyltetrahydropteridin in 120 mM cence (4, 5) should contain the enzyme. Consistent with the dithiothreitol, 66 units of catalase in 10 Al, 10,l of 1 mM surmise are biochemical data demonstrating the presence of ferrous ammonium sulfate, and 20 Al of 150 ,M L-[1- high levels of tryptophan hydroxylase activity in brain re- 14C]tryptophan. The mixture was incubated for 30 min at gions containing the cell bodies of 5HT neurons (3) or richly 370, and 20 ,l (30 nmol/min) of aromatic L-amino-acid de- innervated by their processes (3). It has also been proposed, carboxylase and 40 Al of 2.5 mM pyridoxal-5'-phosphate on indirect evidence, that tryptophan hydroxylase exists in were added. The assay tube was then covered by a rubber different forms, differing either in susceptibility to inhibi- vial cap (Kontes Glass Co.) in which was suspended a plastic tion by drugs (6-8) or possibly, in their relationship to sub- well containing a folded filter paper wick and 250 ,l of NCS cellular organelles (3). Tissue Solubilizer. After an additional 30 min of incubation Direct information of the biochemical structure and re- at 37', 100 Ml of 30% perchloric acid was injected through gional and subcellular localization of the enzyme has been the rubber cap and 14CO2 was trapped at 37° for 90 min. restricted by limited success (9, 10) in obtaining purified The plastic well was removed and the radioactivity was de- tryptophan hydroxylase suitable for the development of spe- termined in 10 ml of Bray's solution by liquid scintillation cific antibodies for immunohistochemistry. Recently the de- spectrometry. Activity was expressed as nmol of CO2 velopment in our laboratory of techniques for the purifica- formed per 60 min. One unit of tryptophan hydroxylase ac- tion and immunohistochemical localization of tyrosine hy- tivity is defined as that amount of enzyme which produces 1 nmol of CO2 per 60 min. Protein was determined by the method of Lowry et Abbreviation: 5HT, serotonin. al. (18), except in monitoring column Requests for reprints and all correspondence to: Dr. Tong H. Joh, effluents, when the absorbance at 280 nm was taken as a Department of Neurology, Laboratory of Neurobiology, Cornell measure of protein concentration. University Medical College, 1300 York Avenue, New York, N.Y. Electrophoresis. Polyacrylamide disc gel electrophoresis 10021. was carried out by the method of Davis (19) and Ornstein 3575 Downloaded by guest on October 3, 2021 3576 Cell Biology: joh et al. Proc. Nat. Acad. Sci. USA 72 (1975) (20) using 7% gel, 0.4 M glycine-Tris buffer, pH 8.2 contain- mM dithiothreitol using a Polytron tissue homogenizer. ing 2 mM dithiothreitol, and 3 mA per tube. Amido black The same buffer was used for Sepharose and Sephadex was used to stain the protein on the gel. To assay enzyme ac- column chromatography. The homogenate was centrifuged tivity of different bands, gels were sliced into pieces of 2 at 39,000 X g for 30 min, and powdered ammonium sulfate mm length and placed in 250 ,l of 0.3 M Tris-acetate buff- was added to the supernatant to 80% saturation. The enzyme er, pH 7.0 containing 2 mM dithiothreitol to elute enzyme. was further purified by three sequential chromatographic The tubes were allowed to stand overnight at 40, and the en- steps: In the first step, the enzyme was passed through a zyme activity was determined in the buffer after the gel Sepharose 4B column achieving 2.8-fold purification of en- slices were removed. zyme and 80% recovery of activity. This step removed, in Production of Antibodies. Purified tryptophan hydroxyl- the void volume, an enzymatically inactive peak containing ase was subjected to polyacrylamide disc gel electrophoresis. substances of high molecular weight and having a high spec- Segments containing active enzyme were pooled from 24 trophotometric reading at 260 nm, indicating the presence gels and homogenized in a glass tube with 2.0 ml of 0.9% so- of compounds with a relatively high content of nucleic dium chloride. An equal volume of complete Freund's adju- acids. If this fraction was not removed, subsequent purifica- vant was added; the mixture was thoroughly emulsified and tion procedures were ineffective because the enzyme would injected subcutaneously into white New Zealand rabbits. aggregate into a high-molecular-weight form which was The injections were repeated every 3 weeks for a period of 9 then inseparable from other proteins by chromatography or weeks. The rabbits were bled 5 days after the final injection. electrophoresis. Immunoglobulin (IgG) was precipitated from the serum at Active fractions were then passed through a hydroxyapa- 50% saturation with ammonium sulfate. Immunodiffusion tite column by stepwise elution of protein using 0.0625, and immunoelectrophoresis were carried out by the methods 0.125, and 0.25 M potassium phosphate buffer, pH 6.8, con- of Ouchterlony (21) and Scheidegger (22), respectively. taining 2 mM dithiothreitol. The highest specific activity Immunohistochemical Localization of Tryptophan Hy- was obtained by elution with 0.125 M buffer. The final step droxylase By Light and Electron Microscopy. Tryptophan consisted of Sephadex G-200 column chromatography in hydroxylase was localized immunohistochemically in rat which a 2.4-fold purification was obtained. The specific ac- brain by use of the peroxidase-antiperoxidase method of tivity of the purified enzyme was 45 units/mg of protein, Sternberger et al. (23). The techniques were identical to which represented a 79.2-fold purification of the enzyme those used by us for localization of tyrosine hydroxylase, and from the 39,000 X g supernatant. the methods are extensively detailed elsewhere (13, 15). After each chromatographic step the enzyme was subject- In summary, rats were anesthetized with pentobarbital ed to polyacrylamide gel electrophoresis. The number of (50 mg/kg) and perfused through the aorta with either 4% protein bands detected on the gels after each step was not paraformaldehyde in phosphate buffer, pH 7.3, or with di- substantially reduced despite an increase in specific activity.
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