146
Brahl Red,earth, ~:.; , i 9'75~ t 4(~- }: l © Elsevier Scientific Publishing Company, Amsterdam -- Printed in ~hc Netherlands
Different forms of tyrosine hydroxylase in central dopaminergic and noradrenergic neurons and sympathetic ganglia
TONG HYUB JOH AND DONALD J. REIS Laboratory of Neurobiology, Department of Nem'ology, Cornell University Medical ~ollege, New York, N.Y. 10021 (U.S.A.)
(Accepted November Ist, 1974)
The enzyme, tyrosine hydroxylase (TH, EC 1.14.16.3) catalyzes the rate limiting step in the biosynthesis of catecholaminesL Because of difficulties in isolating the enzyme, the possibility that T H might exist in different molecular forms has never been examined before. The recent development in our laboratory of methods for purifying T H and producing a specific antibody to it a, has led us to examine the enzyme in different tissues by chromatographic and immunochemical techniques. Male Sprague-Dawley rats were killed by cervical dislocation and superior ce,:vical ganglia, adrenal glands and brain rapidly removed. The brain was dissected into the areas of the locus coeruleus, substantia nigra, the caudate nucleus and hypothalamus as previously described la. Different tissues from t5-30 rats were pooled and homogenized in 2.0 ml of 10 m M potassium phosphate buffer, p H 6.0 and centrifuged for 10 min at 10,000 7.: g. The pellets were homogenized in 1.0 ml of the same buffer and the supernatant added to the supernatant from the initial centrifugation. Approximately 1.5 ml of the final supernatant was put on a Sephadex G150 (superfine grade) column (1.5 c m x 27 cm) which was equilibrated with the homogenizing buffer. Proteins were eluted with the same buffer and collected in 0.65 ml fractions. The fractions were read at 280 and 260 nm in a Gilford Spectrophotoraeter, Model 240, and T H activity of each fraction was assayed by a modification of the method of Coyle 2 using [aaC]tyrosine. The approximate molecular weights of different forms of T H were estimated by the method o f Laurent and Killander 4, calculating Kay based on the Kay of the standard proteins whose Stokes radii are knowna: * For immunochemical studies, T H was purified from bovine adrenal medulla by trypsination of I00,000 ;4 g pellet of the homogenate 8 and specific antibody to T H was produced by the method described in our earlier publication a. The antibody for TH, which was produced in rabbits a, was judged to be specific for the following criteria: (a) immunoelectrophoresis and double diffusion of" anti-
* An assumption was made that TH in different fractions has similar molecular configuration. The MW of TH from bovine adrenal, which was estimated by calculating Kay in the present study, was exactly the same as that obtained by Musacchio et al.~.
147
body run against either partially purified TH from bovine or rat adrenal medulla respectively, yielded a single by the methods of Scheidegger12 and 0uchterlony7, precipitin arc; (b) no precipitin arcs formed when the antibody was run against other catecholamine-synthesizing
enzymes
including dopa decarboxylase ethanolamine-N-methyltransferase
purified
from the bovine adrenal medulla,
(DDC), dopamine-P-hydroxylase (PNMT); (c) the antibody
(DBH), or phenylinhibited TH activity
in purified or crude bovine adrenal extracts as well as TH in homogenates nal gland, superior inhibit
the activities
(e) the antibody contain
cervical ganglia, or specific brain regions; of DBH,
DDC,
was localized
enzyme (c1.g. adrenal
or PNMT
from bovine
immunohistochemically
medulla,
sympathetic
or rat adrenal
only
ganglion
of rat adre-
(d) the antibody in tissues
did not glands ;
known
to
cells and catecholamine-
containing neurons of brain)s. For immunotitration l-6 ~1 of specific and highly cross-reactive antibody to bovine adrenal medulla were added to 50 ~1 of enzymatically active fractions eluted from the Sephadex column or to supernatant of tissue homogenates. Sufficient amounts of control serum were added to each tube to bring the final volume to 60 ,ul. The mixture was allowed to stand for 60 min at room temperature with occasional shaking and then centrifuged at 10,000 x g for 10 min to remove the antigen-antibody complex. TH activity was assayed in a 50 ,ul aliquot of the supernatant. In some experiments tissues were treated with RNase or DNase to determine if nucleic acids or nucleotides were associated with the enzyme. In these studies 2.0 mg of pancreatic RNase (RASE form, Worthington Biochemical Corp.), which is entirely free of protectolytic activity, was added to 1.5 ml of 10,000 x g supernatant of tissue homogenate, and the pH was adjusted to 5.7. The mixture was incubated for 5 min at 37 C and the precipitate was removed by centrifugation at 10,000 x g for 10 min. The supernatant titration.
was used either for Sephadex
column
chromatography
or immuno-
When homogenates of different brain regions or of superior cervical ganglia were passed through a Sephadex Gl50 column, TH activity was eluted in different fractions specific for each tissue (Fig. I). Enzyme activity was detected in 3 distinctive peaks (Fig. I) whose corresponding molecular weights (MWs) were estimated by calibration of the column with standard proteins (Fig. 1). TH of different MWs will be referred to as MW forms of the enzyme. The locus coeruleus (Fig. 1, LC), a region of brain containing cell bodies of noradrenergic neurons13, and the hypothalamus (Fig. I, Hypo) in which TH is primarily contained in the axon terminals of noradrenergic neurons originating, in part, from cell bodies of the locus coeruleusll, contained a high MW form of TH (MW 2: 200,000). Peripheral noradrenergic neurons in the superior cervical ganglion contained an intermediate MW form (MW E 130,000) (Fig. 1, SCG). The 10~ MW form of the enzyme (MW 2: 65,000) was detected in homogenates of the substantia nigra (Fig. 1, SNZ) and was the dominant peak of the caudate nucleus (Fig. 1, CN). The substantia nigra and caudate, respectively, contain cell bodies and terminals of central dopaminergic neurons 13. The substantia nigra also contained some TH of the high MW form (Fig. 1, SNr) and the broad
shoulder
of the TH activity
eluted
148 CN LC 1.0~-
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30
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Fig. I. Sephadex column chromatography of TH from different tissues of rat. 1.5 ml of tissue homogenate was passed over a Sephadex G150 (superfine grade) column (1.5 cm x 27 cm) and collected in fractions of 0.65 ml. Location of fractions in which standard proteins were eluted are indicated on the abscissa of the lower figure. Open circles on each line represent points at which the ratio of the spectrophotometric readings of 280/260 nm were 1.0. See text for details. Abbreviations : BSA, bovine serum albumin; CN, caudate nucleus; HTG, human gamma-globulin; Hypo, hypothalamus; LC, locus coeruleus; SCG, superior cervical ganglion; SN, substantia nigra ; Vo, void volume.
from caudate (Fig. 1, CN) suggests the presence of some TH of high MW in this nucleus. The fractions containing the high MW form of TH had higher spectrophotometric readings at 260 nm than fractions with other MW forms (Fig. 11. Since a high 260 nm reading reflects a high content of nucleic acids, this finding suggests that the high M W form of TH is associated with relatively high concentrations of nucleic acids or nucleotides. We next sought to determine, by immunological methods, whether the different MW forms of TH also differed antigenicatly. In most respects the antigenicity of the different forms of TH appeared similar. For example, no evident differences could be detected between the low and high MW forms of TH from caudate nucleus or locus coeruleus by immunoelectrophoresis, gel diffusion, or by inhibition of enzyme activity with high titers of the specific antibody. However. the shape of the immunotitration curve of the high M W form produced by adding increasing amounts of antibody to active fractions eluted from the Sephadex column differed from that of other M W forms of TH. As illustrated in Fig. 2. the high MW forms of TH from locus coeruleus (Fig. 2, LC), hypothalamus (Fig. 2. Hypo) and substantia nigra (Fig. 2, SNa) had biphasic immunotitration curves. Intermediate or low MW forms from superior cervical ganglion (Fig. 2, SCG), caudate nucleus (Fig. 2. CN) and substantia nigra (Fig. 2, SN2) had monophasic curves.
149
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i
,
0
,
I
3
,
,
I
6
I, , I , , I 0 3 6 Volume antiserum added (pl)
,,I,,I 3
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Fig. 2. Immunotitration by a specific TH antibody of enzyme in eluates of different brain regions and superior cervical ganglion previously separated by Sephadex column chromatography. Aliquots (50 #1) of active fractions from Sephadex column (Fig. 1) were incubated with increasing volumes of specific antibody, centrifuged and enzyme activity remaining in the supernatant measured. Note biphasic and monophasic curves characteristic for different tissues. Abbreviations as in Fig. 1. SNx and SN2 represent different peaks of TH activity in substantia nigra as illustrated in Fig. I. To determine if nucleic acids associated with the high M W form of the enzyme contributed to the M W and was responsible for the biphasic form of the immunotitration curve characteristic of this form of TH, homogenates of the locus coeruleus were incubated with either RNase or DNase and then passed over a Sephadex column and eluted (Fig. 3A). Incubation of homogenates of locus coeruleus with RNase prior to chromatography resulted in: (a) reduction of the M W of T H from ~ 200,000 to _~ 150,000 (Fig. 3A); (b) an increase in the 280/260 nm ratio from 0.89 to 1.42, a change compatible with cleavage of nucleotides; (c) conversion of the biphasic to a monophasic immunotitration curve (Fig. 3B). Preincubation with DNase did not alter the MW, 280/260 nm ratio, nor the immunotitration patterns. Our findings, therefore, suggest that T H exists in several M W forms differing between sympathetic ganglia, central dopaminergic and central noradrenergic neurons; that each M W form of TH consists of the same antigenic enzyme protein unit; and that the high M W form of TH may be associated with an RNA-containing moiety. It is not possible to state if the R N A moiety is naturally linked to T H in situ or becomes attached during the process of homogenization and is hence extrinsic. It seems probable, however, for several reasons, that the R N A moiety associated with the high M W form of T H represents enzyme in a state normally existing in tissue and not an artifact resulting from binding of T H to R N A liberated from other cellular compartments during homogenization. The reasons for this conclusion are these: first, the fact that both high and low M W forms of T H can be eluted from the homogenate of a single brain region or tissue, e.g. substantia nigra or caudate nucleus, seems unlikely to be a consequence of binding of an extraneous R N A macromolecule to only one form of T H ; second, the observations that cell bodies and terminals of
150
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~
1 ~ =- 4 ~I T ~ 2~
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LChomogenate+RNase
7,
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Fig. 3. Effect of ribonuclease (RNase) on TH in locus coeruleus (LC). Pancreatic ribonuclease A (2.0 mg) was added to 1.5 ml of an homogenate of LC, and the pH adjusted to 5.7 at room temper a• ture. The mixture was incubated at 37 °C for 5 min, centrifuged, and the supernatant run through the column as described in Fig. 1. A: Sephadex column chromatography. Note shift of LC peak after RNase. B: immunotitration of TH in bomogenate of LC before and after treatment with RNase. Note that RNase changed the immunotitration curve from a biphasic to a monophasic one.
central noradrenergic neurons (which are located in distinct areas) both contain TH of the high M W form while cell bodies and terminals of dopaminergic neurons (also situated in different regions) contain the low M W form is difficult to reconcile with a chance-mixing hypothesis; finally, the observation that RNase treatment of T H in the locus coeruleus reduced the M W suggests that the RNA associated moiety contributed to the high MW form of TH. However, even if the RNA association is artifactual, the simple fact that only T H from noradrenergic neurons binds nucleotides indicates a difference in this form of the enzyme from T H in dopaminergic and sympathetic neurons. The different MW forms of TH which are separated by chromatography may reflect different states of polymerization or aggregation o f the primary enzyme unit. It has been proposed 6 that the catalytically active fragment of trypsin-digested adrenomedullary T H has a MW o f 34,000. It is of interest that the MW of this fragment is approximately half that of the low MW form of T H in dopaminergic neurons and one-quarter of the MW of the intermediate form of T H characteristic of superior cervical ganglion. On the assumption that the trypsin-digested 34,0130 M W unit is a monomer 6, T H in the caudate may be a dimeric and that of the superior cervical ganglion a tetrameric form of the enzyme. The high MW form of T H found in central noradrenergic neurons, on the other hand, may represent an intermediate (tetrameric) MW form associated with an RNA-moiety of an approximate MW of 60;000. It remains to be established if the MW forms of T H characterizing different
151 types of catecholamine neurons are 'fixed' in life or represent a continuum of MW forms, each in equilibrium with the other, but having, in each type of neuron, a favored state of association. However, the finding of differences in the MW of TH in noradrenergic and dopaminergic neurons may underlie some differences in the molecular biology of these two catecholamine systems of brain including the recently described differences in the capacity of these neurons to accumulate TH in response to reserpine t° and in the intensity and form of immunohistochemical staining of these neuronal systems by labeled antibodies to the enzyme 9.
1 ACKtRS,G. K., Molecular exclusion and restricted diffusion process in molecular-sieve chromatography, Biochemistry, 3 (1964) 723-730. 2 COYLE,J, T., Yyrosine hydroxylase in rat brain--cofactor requirements, regional and subcellular distribution, Bioehem. Pharmacol., 21 (1972) 1935-1944. 3 Jo•, T. H., GEGHMAN, C., AND REIS, D.J., Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2767-2771. 4 LAURENT,T. C., AND K1LLANDER,J., A theory of gel filtration and its experimental verification, J. Chromatogr., 14 (1964) 317-330. 5 LEVITT, M., SPECTOR, S., SJOERDSMA,A., AND UDEYFR1E~D,S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in perfused guinea pig heart, J. Pharmacol., 148 (1965) 1 8. 6 MUSACCHIO,J. M., WURZBURGER,R. J., AND D'ANGELO, G. L., Different molecular forms of bovine adrenal tyrosine hydroxylase, Molec. Pharmacol., 7 (1971) 136-146. 70UCHTERLONY, O , Diffusion-in-gel rnethods for immnunological analysis. In P. KALLOS (Ed.), Progress in Allergy, Vol. V, Karger, Basel, 1958, pp. 1-78. 8 PETRACK, B., SHEPPY, F., AND FETZER, V., Studies on tyrosine hydroxylase from bovine adrenal medulla, J. biol. Chem., 243 (1968) 743 748. 9 PICKEL, V. M., JOH, T. H., FIELD, P. M., BECKER,C. G., AND REIS, D. J., Cellular localization of tyrosine hydroxylase by immunohistochemistry, J. Cytochem. Histochem., (1974) in press. 10 RHs, D. J., JOH, T. H., Ross, R. A., AND PrCKEL, V. M., Reserpine selectively increases tyrosine hydroxylase and dopamine-/3-hydroxylase enzyme protein in central noradrenergic neurons, Brain Research, 81 (1974) 380-386. I I Ross, R. A., AND REIS, D. J., Effects of lesions of locus coeruleus on regional distribution of dopamine-//-hydroxylase activity in rat brain, Brain Research, 73 (1974) 161-166. 12 SCHEIDEG(IER,J. J., Une micro-m6thode de l'immunoelectrophor6se, bit. Arch. Allergy, 7 (1955) 103-110. 13 UNC,ERSTEDT, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., 82, Suppl. 367 (1971) 1-48.