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Effect of ascorbic acid on tyrosine hydroxylase activity in vivo
ARCHIVES
OF
Effect
BIOCHEMISTRY
AND
of Ascorbic YOKO
BIOPHYSICS
Acid
NAKASHIMA,
162, 515-520 (1972)
on Tyrosine
Hydroxylase
RYOKUERO SUZUE, SHOJI KAWADA
Activity
HIROO
in vivo
SANADA,
AND
Nalional
Institute
of Nutrition,
1 Toyamacho,
Shinjuku-ku,
Tokyo 168, Japan
Received November 11, 1971; accepted June 13, 1972 The activity of tyrosine hydroxylase in the homogenate of adrenal gland decreased in scurvy, and it was recovered by the administration of ascorbic acid. The mechanism of increase in tyrosine hydroxylase activity by administration of ascorbic acid has been studied. The enzyme activities of the adrenal homogenates in nonscorbutic and scorbutic guinea pigs were changed neither by dialysis nor by gel filtration on Sephadex G-25. However, stimulation of enzyme activity by the administration of ascorbic acid, was blocked either by puromycin or by actinomycin 1). Tyrosine hydroxylase was purified by ammonium sulfate fractionation, Sephadex G-200, and hydroxylapatite
chromatography.
Antibody
to the partially
purified
enzyme was prepared in rabbit. Immunochemical analysis indicated that there was a constant amount of immunochemically precipitable enzyme per unit of enzyme activity. The studies reported here showed that the increase of enzyme activity by the administration of ascorbic acid was due to the increased amount of the enzyme pro-
tein. There are some reports on changes of enzyme activity involved in biological oxidations b;y administration of ascorbic acid. Ascorbic acid is known to have an essential function in (i) the hydroxylation of proline in collagen formation (l), (ii) the biogenesis of corticosteroid in the adrenals (a), (iii) the hydroxylation of aromat,ic compounds in the liver (3), and also (iv) a cofactor of dopamine-p-hydroxylase (4). However, the mechanism by which ascorbic acid exerts its function is not clarified yet. As shown in the previous publication (5), the activit’y of tyrosine hydroxylase decreased in the scorbutic guinea pigs, and was recovered by the administration of ascorbic acid. In order to clarify this mechanism of increase in tyrosine hydroxylase activity of adrenal gland, we performed experiments using inhibitors of protein synthesis and immunochemical analysis. When puromycin or actinomycin D was given to scorbutic guinea pigs, no rise of tyrosine hydroxylase activity was observed upon administration
of ascorbic acid. Furthermore, immunochemical analysis indicated that the increase of the tyrosine hydroxylase activity in upon administration of adrenal gland ascorbic acid was due to a specific increase of tyrosine hydroxylase enzyme protein. MATERIALS
METHODS
Materials Puromycin was purchased from Nutritional Biochemicals Corporation. Actinomycin D was obtained from Mann Research Laboratories, Inc. Complete Freund’s adjuvant was purchased from Itaron Laboratories. All other chemicals used were analytical grade. Animals ilscorbic acid-deficient diet was prepared as described by Nakashima et aZ. (5). Male guinea pigs, weighing about, 300 g, were fed ascorbic acid-deficient diet for 18 days. The animals were separated into two groups. One group was given 200 mg of ascorbic acid in a volume of 2 ml per day by intraperitoneal injection for 3 days before sacrifice (nonscorbutic group). The other group was given the same volume of 0.9% NaCl 515
Copyright 0 1972 by Academic Pwss, Inc. All rights of reproduction in any form reserved.
AND
516
NAKASHIMA
solution (scorbutic group). These animals were pair fed. Groups of guinea pigs were treated with puromycin (25 mg/ml) or actinomycin D (300 rg/ml) dissolved in saline solution. In the experiments in which RNA or protein synthesis inhibitors were used, 25 mg of puromycin were administered four times at int,ervals of 6 hr by intraperitoneal injection (total 100 mg per animal), and 300 rg of actinomycin D per animal per day were given for 2 days intraperitoneally. Assay
of Tyrosine
Hydroxylase
and Protein
The enzyme activity was assayed according to Nagatsu et al. (6) as modified by Nakashima et al. (5). Protein was assayed by the method of Lowry et al. (7), or by the method of Warburg and Christian (8). Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [aH]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg protein. Dialysis raphy
and Sephadex
G-25 Column
Chromatog-
The adrenal glands were homogenized with 3 vol of ice-cold 0.32 M sucrose using a PotterElvehjem homogenizer at 0°C. The adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were separately dialyzed against distilled water for 24 hr. The dialyzate of the nonscorbutic guinea pigs was evaporated at 30°C in vacua to half of the original volume and used as a concentrated dialyzate. The tyrosine hydroxylase activities of the original and of dialyzed homogenates were determined. The 105,OOOg supernatant fraction of the adrenal gland from the nonscorbutic or the scorbutic guinea pig was applied to a Sephadex G-25 column (20 X 1 cm) and the large molecular weight fraction was collected. The enzyme activity of the fraction was assayed. Purijcation
of Tyrosine
ET AL. stand for 60 min and was then centrifuged for 30 min at 20,OOOg. The supernatant fluid (27 ml) was brought to 457c saturation with solid ammonium sulfate (3.4 g), stirred, and the pH of the solution was adjusted to 7.0. This solution was allowed to stand for 60 min. The precipitate was collected by centrifugation at 20,OOOgfor 30 min, suspended in a minimum volume of 0.02 M phosphate buffer (pH 6.5), and dialyzed overnight against the same buffer. The dialyzed enzyme was applied to a column of hydroxylapatite (100 X 1 cm) equilibrated with 0.02 M potassium phosphate (pH 6.0). Elution was accomplished with a 200-ml linear potassium phosphate gradient (0.02-0.1 M, pH 6.5). The flow rate was 5 ml per hr. The effluent was collected fractionally and the fractions were assayed for tyrosine hydroxylase activity. The most active fractions were pooled and concentrated with collodion bag in vacua. The enzyme was applied to a Sephadex G-200 column (100 X 1 cm) equilibrated with 0.02 M potassium phosphate (pH 6.0). The flow rate was 10 ml per hr and the effluent was collected fractionally. The enzyme activity of the fractions was determined. The most active fractions were collected and concentrated with collodion bag in VUCUO.The purity of the enzyme was checked by polyacrylamide gel electrophoresis and the enzyme was about 70yo pure. The results are summarized in Table I. Preparation
of Antiserum
For the preparation of an antiserum specific for tyrosine hydroxylase, the purified enzyme (sp act 497.3 nmoles/mg protein) was injected according to the following regimen. Initially, 2 ml of the purified enzyme solution containing 1.5 TABLE
I
PURIFICATION OF TYROSINE HYDROXYLASE ADRENAL GLAND OF GUINEA PIG
OF
Hydroxylase
Thirty male guinea pigs weighing 400-500 g were decapitated and the adrenal glands were quickly removed. Nine grams of the adrenal glands were homogenized in 3 vol of 0.32 M sucrose. The homogenate was centrifuged for 60 min at 105,OOOgin a Spinco model L-2 centrifuge. The precipitate was discarded and 30 ml of the supernatant fraction were used for the purification of tyrosine hydroxylase. To give a 25yo saturation, 4.3 g of solid ammonium sulfate were added slowly to the supernatant solution of adrenal homogenate which was being stirred while the pH was maintained at 7.0 by adding 2% ammonium hydroxide. This solution was allowed to
Step
Protein (4
Total activity
Homogenate 105,000 g supernatant Ammonium sulfate (25-45s) Hydroxylapatite Sephadex G-200
1831 780
6225.4 6084.0
3.4 7.8
298
4380.6
14.7
2331.7 1591.4
153.4 497.3
15.2 3.2
Specific activity”
a Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [aH]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg of protein.
EFFECT
OF ASCORBIC
ACID
mg of protein per ml were homogenized with an equal volume of complete Freund’s adjuvant. This mixture was injected intramuscularly on the dorsal region of a rabbit. Two and 4 weeks later, 1 mg of the enzyme was injected at a time in the same rabbit. The rabbit was bled every week and the titer of each serum sample was determined. Highest titer was obtained 7 weeks after the first antigen injection and serum from immunized rabbit was pooled. Immunochemical use
Titration
of Tyrosine
Hydroxyl-
As it was found that almost all of the total activity was recovered in the 105,000~ supernatant solution, the supernatant fractions of adrenal glands of the nonscorbutic and scorbutic guinea pigs were used to determine the amount of enzyme protein. A constant amount of antiserum (0.5 ml) was added to increasing amounts of supernatant fluid of the nonscorbutic and scorbutic guinea pigs, previously assayed for tyrosine hydroxylase activity. The mixture was diluted to a final volume of 1.6 ml with 0.9% NaCl and stored overnight at 4°C. The tyrosine hydroxylase-antibody precipitates were removed by cenand the tyrosine hydroxylase actrifugation, tivities in the supernatant solution were estimated. A supernatant fraction (0.8 ml) was added to a standard reaction mixture, and it was incubated at 37°C for 30 min. The reaction was terminated by the addition of 2.0 ml of 1% picric
acid. After centrifugation,
the supernatant
517
HYDROXYLASE
weight activator. The adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were dialyzed and the low molecular weight materials were removed. The enzyme activities of dialyzed homogenates were compared with those of the nondialyzed homogenates (Table III). In both animals, TABLE
II
EFFECT OF ASCORBIC ACID ON TYROSINE HYDROXYLASE ACTIVITY IN ADRENAL GLAND OF GUINEA PIG Nonscorbutic Specific activitya Total activityb
Scorbutic
3.5 f
0.2‘
1.8 f
133.9 i
2.6
77.0 f
0.3 14.0
(1Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [3H]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg of protein. b Total activity was defined as the activity of the enzyme which catalyzed the release of 3H from [3H]tyrosine in 30 min per whole tissue. L Values represent the mean of nine guinea pigs f SE. Tyrosine hydroxylase activity was assayed in homogenates of adrenal glands from the nonscorbutic and scorbutic guinea pigs.
solu-
tion was passed through a column of layering Dowex 2-W (0.5 X 3 cm) to absorb picric acid before assaying for released tritium. RESULTS
Effect of Ascorbic Acid on droxylase Activity
ON TYROSINE
Tyrosine Hy-
Tyrosine hydroxylase activity in the adrenal whole homogenate of the nonscorbutic guinea pig was compared with that of the scorbutic guinea pig (Table II). The enzyme activity of the nonscorbutic guinea pig was a-fold higher than that of the scorbutic guinea pig in the adrenal gland.
Effect of Macromolecular Fraction (Protein Fraction) and Low Molecular Weight Fraction on Tyrosine Hydroxylase Activity The possibility was examined that the low tyrosine hydroxylase activity of scorbutic guinea pig is due to loss of a low molecular
TABLE
III
EFFECT OF DIALYSIS ON TYROSINE HYDROXYLME ACTIVITY IN ADRENAL GL.~ND OF THE NONSCORBUTIC AND SCORBUTIC GUINEA PIGS Animals
Nonscorbutic Scorbutic
Nondialyzed homogenate* (sp act)
Dialyzed homogenate (SP act)
3.83
3.42
1.77
1.66
Scorbutic dialyzed homogenate + concentrated nonscorbutic dialyzate
1.83
Q Adrenal homogenate of the nonscorbutic and scorbutic guinea pigs were dialyzed in a cellophane tube at 4’C against distilled water for 24 hr. Tyrosine hydroxylase activity was determined as described under Method. Dialyzate of the nonscorbutic guinea pig was concentrated to 1 ml; 0.1 ml of the concentrated dialyzate was added to the incubation medium. All values were expressed as nmoles per mg protein per 30 min.
518
NAKASHIMA TABLE
ET AL.
IV
EFFECT OF GEL FILTRATION ON TYROSINE HYDROXYLASE ACTIVITY IN ADRENAL GLANDS OF NONSCORBUTIC AND SCORBUTIC GUINEA PIG Animals
Nonscorbutic” Scorbutic
105,000g supernatant (sp act)
Protein fraction (SP act)
9.08 4.95
5.05
9.12
0 Adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were centrifuged at 105,000 TVfor 45 min and the supernatant solution was separated. Half of the 105,000 q supernatant solution was applied to a column of Sephadex G-25 (1.0 X 10 cm), and the protein fractions were collected. The assays of tyrosine hydroxylase activity and protein were carried out as described under Methods. All values were expressed as nmoles per mg protein per 30 min.
no remarkable difference on the enzyme activity was observed between the dialyzed and nondialyzed homogenates. When concentrated dialyzate of the nonscorbutic guinea pig was added to the dialyzed homogenate of the scorbutic guinea pig, no stimulation of the activity was observed. The 105,OOOg supernatant fractions of the nonscorbutic and scorbutic guinea pigs were applied t’o a Sephadex G-25 column (20 X 1.0 cm), and the protein fractions were compared with t,hose of the original supernatants. In both the scorbutic and nonscorbutic animals, t,he specific activities of the protein fractions revealed no difference from that of the original supernatant fractions. As Fez+, DMPH4, and mercaptoethanol were required for maximum activity of tyrosine hydroxylase, sufhcient amount of them were added to the reaction mixture. From these results, it was concluded that the difference of tyrosine hydroxylase activit’ies in the scorbutic and nonscorbutic guinea pigs did not depend on low molecular weight materials. Effect of Puromycin and Actinomycin Tyrosine Hydroxylase Activity
D on
Puromycin and actinomycin D were administered to determine whether the rise of tyrosine hydroxylase activity was due to an increase of enzyme protein synthesis. As shown in Fig. 1, a significant increase in
a+ 0 ASCORBIC PUROMYCIN
L
ACID
+
+
-
-
+
-
FIG. 1. Effect of puromycin on the response of adrenal tyrosine hydroxylase activity to ascorbic acid. Male guinea pigs were fed an ascorbic aciddeficient diet for 2 weeks, and separated into three groups. Two hundred milligrams of ascorbic acid were given to two groups per day by intraperit.oneal injection. To one of the two groups, 25 mg of puromycin were administered 30 min before the ascorbic acid injection. The administration of puromycin was repeated four times at intervals of 6 hr. They were sacrificed 24 hr later. Tyrosine hydroxylase was assayed in adrenal homogenates. Values represent the mean for three guinea pigs f SE. This group was fed a normal diet.
the enzyme activity was found 24 hr after the administration of ascorbic acid. However, puromycin blocked the increase in the enzyme activity by ascorbic acid. Tyrosine hydroxylase activities of the ascorbic acidadminist,ered guinea pigs were found to be l&fold higher in 24 hr (Fig. l), and in 48 hr 3.0-fold higher than the values found in the scorbutic guinea pigs (Fig. 2). As shown in Fig. 2, the administration of actinomycin D did not alter the activity of tyrosine hydroxylase. However, actinomycin D prevented the increase in enzyme activity in the scorbutic guinea pig caused by ascorbic acid. These results suggested that the
EFFECT
OF ASCORBIC
ACID
ASCORBIC
ACID
ACTINOMYCIN
0
+
t
-
-
-
+
-
t
2. Effect of actinomycin D on the response of adrenal tyrosine hydroxylase activity to ascorbic acid. Male guinea pigs were fed an ascorbic acid-deficient diet for 2 weeks and separated into three groups. Two of them were given 200 mg of ascorbic acid per day by intraperitoneal injection. To one of the ascorbic acid administration groups, 300 Hg of actinomycin D were administered 30 min before the ascorbic acid injection. This treatment was repeated again after 24 hr. They were sacrificed 48 hr later. Tyrosine hydroxylase was assayed in adrenal homogenates. Values represent the mean for three guinea pigs f SE. This group was fed a normal diet. FIG.
ON TYROSINE
519
HYDROXYLASE
immunochemical analysis. The specific activities in lOrj,OOOg supernatant fractions from the nonscorbutic and scorbutic guinea pigs were 3.81 nmoles per mg protein and 1.97 nmoles, respectively. For the experiment shown in Fig. 3, a constant amount of antiserum (0.5 ml) was added to increasing volumes of supernatant fluid of the nonscorbutic and scorbutic guinea pigs. The quantity of supernatant fluid of the nonscorbutic and scorbutic guinea pigs, which completely titrated the same amount of antibody, was 0.09 and 0.18 ml, respectively. ascorbic acid adminisTherefore, after tration, the amount of immunochemically reactive tyrosine hydroxylase increased 2-fold. Thus, the increase in tyrosine hydroxylase activity by the administration of 5.01
administration of ascorbic acid to the scorbutic guinea pigs stimulated protein synthesis involved in tyrosine hydroxylase activity. One possible explanation for these results was that the changes in tyrosine hydroxylase activity that followed ascorbic acid administration were due to changing amounts of enzyme protein. Therefore, we measured the levels of tyrosine hydroxylase protein in the adrenal gland of the guinea pig by immunochemical titration. Tyrosine Hytlroxylase Content of the Nonscorbutic and Scorbutic Guinea Pigs The scorbutic guinea pigs weighing about 300 g were separated into two groups. One group was ad.ministered 200 mg of ascorbic acid per day by intraperitoneal injection (the nonscorbutic group). The other group was given the same volume of 0.9 % NaCl solution (the scorbutic group). The adrenal glands of the nonscorbutic and scorbutic guinea pigs were homogenized and centrifuged for 60 min at 105,OOOg, and the resulting supernatant fluid was used for
SUPERNATANT
(ml)
3. The effect of administration of ascorbic acid on tyrosine hydroxylase content in the adrenal glands of guinea pigs. A constant amount of antiserum (0.5 ml) was added to increasing quantities of 105,000g supernatant solutions of adrenal gland prepared from the nonscorbutic (0) and scorbutic (X) guinea pigs. The tyrosine hydroxylase activity found after removal of the antigen-antibody precipitate is indicated in the ordinate. FIG.
520
NAKASHIMA
ascorbic acid was due to an increase in the amount of enzyme protein. These experiments indicate that the differences in specific activity of tyrosine hydroxylase seen upon ascorbic acid administration are actually due to the different amounts of enzyme in the adrenal glands of these animals. Thus, the change in tyrosine hydroxylase activity in the nonscorbutic and scorbutic guinea pigs can be entirely explained on the basis of changing amount of enzyme. DISCUSSION
In the present paper, it was demonstrated that tyrosine hydroxylase activity in adrenal gland of guinea pig decreased in scurvy, and it was recovered by the administration of ascorbic acid. This alteration might be due to the following causes. (a) Directly, ascorbic acid activated the enzyme. (b) Ascorbic acid affected the activation of low molecular weight coenzyme or metal ion such as Fe2+ (9). (c) Ascorbic acid increased the amount of protein such as cytochrome P-450 (10) involved in the enzyme activation. (d) Ascorbic acid changed the levels of tyrosine hydroxylase in adrenal gland of guinea pig. As indicated in a previous study (5), when 1 X lOW-1 X 1O-4 M ascorbic acid was added to the homogenate of the scorbutic guinea pig, the enzyme activity was not stimulated. Therefore, ascorbic acid did not activate the enzyme directly. Shiman et al. (11) demonstrated that tyrosine hydroxylase was inactivat,ed by Hz02 that was generated during the nonenzymatic oxidation of tetrahydropterin, and could be protected by catalase, peroxidase, or Fez+ from Hz02-mediated inactivation. Sufficient amounts of Fe2+ were added to the react,ion mixture in our experiment. Therefore, it was not considered that the catalase and peroxidase protected the enzyme activity of the nonscorbutic guinea pig and did not protect the enzyme activity of the scorbutic guinea pig from HzOzmediated inactivation. The experiments of gel filtration and enzyme purification were performed using 105,OOOg supernatant fraction. The data concerning the intracellular localization of tyrosine hydroxylase were rather conflicting.
ET AL.
Nagatsu et al. (9) and Pet,rack et al. (14) reported that tyrosine hydroxylase activity of bovine adrenal medulla was associated with particles. Stjarne and Lishajko (15), however, showed that this enzyme was found in a supernatant fraction. In this paper, when the adrenal glands were homogenized by a Potter-Elvehjem homogenizer, tyrosine hydroxylase activity was found in the 105,OOOgsupernatant’ fraction. Therefore, these experiments were performed using supernatant fraction. ACKNOWLEDGMENT The authors express their thanks Potjanee Threeratana (Ramathibody Bangkok, Thailand) for her excellent assistance.
to Miss Hospital, technical
REFERENCES 1. HUTTON, J. J., JR.,TAPPEL, A. L., END UDENFRIEND, S. (1967) Arch. Biochem. Biophys. 118, 231. 2. KERSTEN, H., LEONHAUSTER, S., AND STAUDINGER, H. (1958) Biochim. Biophys. Acta 29, 350. 3. STAUDINGER, H., KHISCH, K., AND LEONHAUSTER, S. (1961) Ann. N. Y. Acad. Sci. 92, 195. 4. LEVIN, E. Y., LEVENBURG, B., AND KAUFMAN, S. (1960) J. Biol. Chem. 236, 2080. 5. NaKASHIM.4, Y., SUZUE, It., SANADA, H., AND KAlvlDA, S. (1970) J. Fitaminol. 16, 276. 6. NAGATSU, T., LEVITT, M., AND UDENFRIEND, S. (1964) Anal. Biochem. 9, 122. 7. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 8. WARBURG, O., AND CHRISTIAN, W. (1936) Biochem. Z. 267, 291. 9. NAGATSU, T., LEVITT, M., AND UDENFRIEND, S. (1964) J. Biol. Chem. 239, 2910. 10. LEVEN, H-W., DEGK~ITZ, E., AND STAUDINGER, H. (1970) 2. PhysioZ. Chem. 361,995. 11. SHIMAN, R., AKINO, M., AND KAUFMAN, S. (1971) J. BioZ. Chem. 246, 1330. 12. TAYLOR, R. J. JR., STUBBS, C. S. JR., AND ELLENBOGEN, L. (1969) Biochem. Pharmacol. 18, 587. 13. KOMETANI, W. (1959) Osaka Daigaku Igaku Zasshi 11, 1855. 14. PETRACK, B., SNEPPY, F., AND FETZER, V. (1968) J. Biol. Chem. 243, 743. 15. STJ;~RNE, L., AND LISHAJKO, F. (1967) Biothem. Pharmacol. 16, 1719.
OF
Effect
BIOCHEMISTRY
AND
of Ascorbic YOKO
BIOPHYSICS
Acid
NAKASHIMA,
162, 515-520 (1972)
on Tyrosine
Hydroxylase
RYOKUERO SUZUE, SHOJI KAWADA
Activity
HIROO
in vivo
SANADA,
AND
Nalional
Institute
of Nutrition,
1 Toyamacho,
Shinjuku-ku,
Tokyo 168, Japan
Received November 11, 1971; accepted June 13, 1972 The activity of tyrosine hydroxylase in the homogenate of adrenal gland decreased in scurvy, and it was recovered by the administration of ascorbic acid. The mechanism of increase in tyrosine hydroxylase activity by administration of ascorbic acid has been studied. The enzyme activities of the adrenal homogenates in nonscorbutic and scorbutic guinea pigs were changed neither by dialysis nor by gel filtration on Sephadex G-25. However, stimulation of enzyme activity by the administration of ascorbic acid, was blocked either by puromycin or by actinomycin 1). Tyrosine hydroxylase was purified by ammonium sulfate fractionation, Sephadex G-200, and hydroxylapatite
chromatography.
Antibody
to the partially
purified
enzyme was prepared in rabbit. Immunochemical analysis indicated that there was a constant amount of immunochemically precipitable enzyme per unit of enzyme activity. The studies reported here showed that the increase of enzyme activity by the administration of ascorbic acid was due to the increased amount of the enzyme pro-
tein. There are some reports on changes of enzyme activity involved in biological oxidations b;y administration of ascorbic acid. Ascorbic acid is known to have an essential function in (i) the hydroxylation of proline in collagen formation (l), (ii) the biogenesis of corticosteroid in the adrenals (a), (iii) the hydroxylation of aromat,ic compounds in the liver (3), and also (iv) a cofactor of dopamine-p-hydroxylase (4). However, the mechanism by which ascorbic acid exerts its function is not clarified yet. As shown in the previous publication (5), the activit’y of tyrosine hydroxylase decreased in the scorbutic guinea pigs, and was recovered by the administration of ascorbic acid. In order to clarify this mechanism of increase in tyrosine hydroxylase activity of adrenal gland, we performed experiments using inhibitors of protein synthesis and immunochemical analysis. When puromycin or actinomycin D was given to scorbutic guinea pigs, no rise of tyrosine hydroxylase activity was observed upon administration
of ascorbic acid. Furthermore, immunochemical analysis indicated that the increase of the tyrosine hydroxylase activity in upon administration of adrenal gland ascorbic acid was due to a specific increase of tyrosine hydroxylase enzyme protein. MATERIALS
METHODS
Materials Puromycin was purchased from Nutritional Biochemicals Corporation. Actinomycin D was obtained from Mann Research Laboratories, Inc. Complete Freund’s adjuvant was purchased from Itaron Laboratories. All other chemicals used were analytical grade. Animals ilscorbic acid-deficient diet was prepared as described by Nakashima et aZ. (5). Male guinea pigs, weighing about, 300 g, were fed ascorbic acid-deficient diet for 18 days. The animals were separated into two groups. One group was given 200 mg of ascorbic acid in a volume of 2 ml per day by intraperitoneal injection for 3 days before sacrifice (nonscorbutic group). The other group was given the same volume of 0.9% NaCl 515
Copyright 0 1972 by Academic Pwss, Inc. All rights of reproduction in any form reserved.
AND
516
NAKASHIMA
solution (scorbutic group). These animals were pair fed. Groups of guinea pigs were treated with puromycin (25 mg/ml) or actinomycin D (300 rg/ml) dissolved in saline solution. In the experiments in which RNA or protein synthesis inhibitors were used, 25 mg of puromycin were administered four times at int,ervals of 6 hr by intraperitoneal injection (total 100 mg per animal), and 300 rg of actinomycin D per animal per day were given for 2 days intraperitoneally. Assay
of Tyrosine
Hydroxylase
and Protein
The enzyme activity was assayed according to Nagatsu et al. (6) as modified by Nakashima et al. (5). Protein was assayed by the method of Lowry et al. (7), or by the method of Warburg and Christian (8). Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [aH]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg protein. Dialysis raphy
and Sephadex
G-25 Column
Chromatog-
The adrenal glands were homogenized with 3 vol of ice-cold 0.32 M sucrose using a PotterElvehjem homogenizer at 0°C. The adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were separately dialyzed against distilled water for 24 hr. The dialyzate of the nonscorbutic guinea pigs was evaporated at 30°C in vacua to half of the original volume and used as a concentrated dialyzate. The tyrosine hydroxylase activities of the original and of dialyzed homogenates were determined. The 105,OOOg supernatant fraction of the adrenal gland from the nonscorbutic or the scorbutic guinea pig was applied to a Sephadex G-25 column (20 X 1 cm) and the large molecular weight fraction was collected. The enzyme activity of the fraction was assayed. Purijcation
of Tyrosine
ET AL. stand for 60 min and was then centrifuged for 30 min at 20,OOOg. The supernatant fluid (27 ml) was brought to 457c saturation with solid ammonium sulfate (3.4 g), stirred, and the pH of the solution was adjusted to 7.0. This solution was allowed to stand for 60 min. The precipitate was collected by centrifugation at 20,OOOgfor 30 min, suspended in a minimum volume of 0.02 M phosphate buffer (pH 6.5), and dialyzed overnight against the same buffer. The dialyzed enzyme was applied to a column of hydroxylapatite (100 X 1 cm) equilibrated with 0.02 M potassium phosphate (pH 6.0). Elution was accomplished with a 200-ml linear potassium phosphate gradient (0.02-0.1 M, pH 6.5). The flow rate was 5 ml per hr. The effluent was collected fractionally and the fractions were assayed for tyrosine hydroxylase activity. The most active fractions were pooled and concentrated with collodion bag in vacua. The enzyme was applied to a Sephadex G-200 column (100 X 1 cm) equilibrated with 0.02 M potassium phosphate (pH 6.0). The flow rate was 10 ml per hr and the effluent was collected fractionally. The enzyme activity of the fractions was determined. The most active fractions were collected and concentrated with collodion bag in VUCUO.The purity of the enzyme was checked by polyacrylamide gel electrophoresis and the enzyme was about 70yo pure. The results are summarized in Table I. Preparation
of Antiserum
For the preparation of an antiserum specific for tyrosine hydroxylase, the purified enzyme (sp act 497.3 nmoles/mg protein) was injected according to the following regimen. Initially, 2 ml of the purified enzyme solution containing 1.5 TABLE
I
PURIFICATION OF TYROSINE HYDROXYLASE ADRENAL GLAND OF GUINEA PIG
OF
Hydroxylase
Thirty male guinea pigs weighing 400-500 g were decapitated and the adrenal glands were quickly removed. Nine grams of the adrenal glands were homogenized in 3 vol of 0.32 M sucrose. The homogenate was centrifuged for 60 min at 105,OOOgin a Spinco model L-2 centrifuge. The precipitate was discarded and 30 ml of the supernatant fraction were used for the purification of tyrosine hydroxylase. To give a 25yo saturation, 4.3 g of solid ammonium sulfate were added slowly to the supernatant solution of adrenal homogenate which was being stirred while the pH was maintained at 7.0 by adding 2% ammonium hydroxide. This solution was allowed to
Step
Protein (4
Total activity
Homogenate 105,000 g supernatant Ammonium sulfate (25-45s) Hydroxylapatite Sephadex G-200
1831 780
6225.4 6084.0
3.4 7.8
298
4380.6
14.7
2331.7 1591.4
153.4 497.3
15.2 3.2
Specific activity”
a Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [aH]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg of protein.
EFFECT
OF ASCORBIC
ACID
mg of protein per ml were homogenized with an equal volume of complete Freund’s adjuvant. This mixture was injected intramuscularly on the dorsal region of a rabbit. Two and 4 weeks later, 1 mg of the enzyme was injected at a time in the same rabbit. The rabbit was bled every week and the titer of each serum sample was determined. Highest titer was obtained 7 weeks after the first antigen injection and serum from immunized rabbit was pooled. Immunochemical use
Titration
of Tyrosine
Hydroxyl-
As it was found that almost all of the total activity was recovered in the 105,000~ supernatant solution, the supernatant fractions of adrenal glands of the nonscorbutic and scorbutic guinea pigs were used to determine the amount of enzyme protein. A constant amount of antiserum (0.5 ml) was added to increasing amounts of supernatant fluid of the nonscorbutic and scorbutic guinea pigs, previously assayed for tyrosine hydroxylase activity. The mixture was diluted to a final volume of 1.6 ml with 0.9% NaCl and stored overnight at 4°C. The tyrosine hydroxylase-antibody precipitates were removed by cenand the tyrosine hydroxylase actrifugation, tivities in the supernatant solution were estimated. A supernatant fraction (0.8 ml) was added to a standard reaction mixture, and it was incubated at 37°C for 30 min. The reaction was terminated by the addition of 2.0 ml of 1% picric
acid. After centrifugation,
the supernatant
517
HYDROXYLASE
weight activator. The adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were dialyzed and the low molecular weight materials were removed. The enzyme activities of dialyzed homogenates were compared with those of the nondialyzed homogenates (Table III). In both animals, TABLE
II
EFFECT OF ASCORBIC ACID ON TYROSINE HYDROXYLASE ACTIVITY IN ADRENAL GLAND OF GUINEA PIG Nonscorbutic Specific activitya Total activityb
Scorbutic
3.5 f
0.2‘
1.8 f
133.9 i
2.6
77.0 f
0.3 14.0
(1Specific activity was defined as the activity of enzyme which catalyzed the release of 3H from [3H]tyrosine equivalent to the formation of 1 nmole of DOPA in 30 min per mg of protein. b Total activity was defined as the activity of the enzyme which catalyzed the release of 3H from [3H]tyrosine in 30 min per whole tissue. L Values represent the mean of nine guinea pigs f SE. Tyrosine hydroxylase activity was assayed in homogenates of adrenal glands from the nonscorbutic and scorbutic guinea pigs.
solu-
tion was passed through a column of layering Dowex 2-W (0.5 X 3 cm) to absorb picric acid before assaying for released tritium. RESULTS
Effect of Ascorbic Acid on droxylase Activity
ON TYROSINE
Tyrosine Hy-
Tyrosine hydroxylase activity in the adrenal whole homogenate of the nonscorbutic guinea pig was compared with that of the scorbutic guinea pig (Table II). The enzyme activity of the nonscorbutic guinea pig was a-fold higher than that of the scorbutic guinea pig in the adrenal gland.
Effect of Macromolecular Fraction (Protein Fraction) and Low Molecular Weight Fraction on Tyrosine Hydroxylase Activity The possibility was examined that the low tyrosine hydroxylase activity of scorbutic guinea pig is due to loss of a low molecular
TABLE
III
EFFECT OF DIALYSIS ON TYROSINE HYDROXYLME ACTIVITY IN ADRENAL GL.~ND OF THE NONSCORBUTIC AND SCORBUTIC GUINEA PIGS Animals
Nonscorbutic Scorbutic
Nondialyzed homogenate* (sp act)
Dialyzed homogenate (SP act)
3.83
3.42
1.77
1.66
Scorbutic dialyzed homogenate + concentrated nonscorbutic dialyzate
1.83
Q Adrenal homogenate of the nonscorbutic and scorbutic guinea pigs were dialyzed in a cellophane tube at 4’C against distilled water for 24 hr. Tyrosine hydroxylase activity was determined as described under Method. Dialyzate of the nonscorbutic guinea pig was concentrated to 1 ml; 0.1 ml of the concentrated dialyzate was added to the incubation medium. All values were expressed as nmoles per mg protein per 30 min.
518
NAKASHIMA TABLE
ET AL.
IV
EFFECT OF GEL FILTRATION ON TYROSINE HYDROXYLASE ACTIVITY IN ADRENAL GLANDS OF NONSCORBUTIC AND SCORBUTIC GUINEA PIG Animals
Nonscorbutic” Scorbutic
105,000g supernatant (sp act)
Protein fraction (SP act)
9.08 4.95
5.05
9.12
0 Adrenal homogenates of the nonscorbutic and scorbutic guinea pigs were centrifuged at 105,000 TVfor 45 min and the supernatant solution was separated. Half of the 105,000 q supernatant solution was applied to a column of Sephadex G-25 (1.0 X 10 cm), and the protein fractions were collected. The assays of tyrosine hydroxylase activity and protein were carried out as described under Methods. All values were expressed as nmoles per mg protein per 30 min.
no remarkable difference on the enzyme activity was observed between the dialyzed and nondialyzed homogenates. When concentrated dialyzate of the nonscorbutic guinea pig was added to the dialyzed homogenate of the scorbutic guinea pig, no stimulation of the activity was observed. The 105,OOOg supernatant fractions of the nonscorbutic and scorbutic guinea pigs were applied t’o a Sephadex G-25 column (20 X 1.0 cm), and the protein fractions were compared with t,hose of the original supernatants. In both the scorbutic and nonscorbutic animals, t,he specific activities of the protein fractions revealed no difference from that of the original supernatant fractions. As Fez+, DMPH4, and mercaptoethanol were required for maximum activity of tyrosine hydroxylase, sufhcient amount of them were added to the reaction mixture. From these results, it was concluded that the difference of tyrosine hydroxylase activit’ies in the scorbutic and nonscorbutic guinea pigs did not depend on low molecular weight materials. Effect of Puromycin and Actinomycin Tyrosine Hydroxylase Activity
D on
Puromycin and actinomycin D were administered to determine whether the rise of tyrosine hydroxylase activity was due to an increase of enzyme protein synthesis. As shown in Fig. 1, a significant increase in
a+ 0 ASCORBIC PUROMYCIN
L
ACID
+
+
-
-
+
-
FIG. 1. Effect of puromycin on the response of adrenal tyrosine hydroxylase activity to ascorbic acid. Male guinea pigs were fed an ascorbic aciddeficient diet for 2 weeks, and separated into three groups. Two hundred milligrams of ascorbic acid were given to two groups per day by intraperit.oneal injection. To one of the two groups, 25 mg of puromycin were administered 30 min before the ascorbic acid injection. The administration of puromycin was repeated four times at intervals of 6 hr. They were sacrificed 24 hr later. Tyrosine hydroxylase was assayed in adrenal homogenates. Values represent the mean for three guinea pigs f SE. This group was fed a normal diet.
the enzyme activity was found 24 hr after the administration of ascorbic acid. However, puromycin blocked the increase in the enzyme activity by ascorbic acid. Tyrosine hydroxylase activities of the ascorbic acidadminist,ered guinea pigs were found to be l&fold higher in 24 hr (Fig. l), and in 48 hr 3.0-fold higher than the values found in the scorbutic guinea pigs (Fig. 2). As shown in Fig. 2, the administration of actinomycin D did not alter the activity of tyrosine hydroxylase. However, actinomycin D prevented the increase in enzyme activity in the scorbutic guinea pig caused by ascorbic acid. These results suggested that the
EFFECT
OF ASCORBIC
ACID
ASCORBIC
ACID
ACTINOMYCIN
0
+
t
-
-
-
+
-
t
2. Effect of actinomycin D on the response of adrenal tyrosine hydroxylase activity to ascorbic acid. Male guinea pigs were fed an ascorbic acid-deficient diet for 2 weeks and separated into three groups. Two of them were given 200 mg of ascorbic acid per day by intraperitoneal injection. To one of the ascorbic acid administration groups, 300 Hg of actinomycin D were administered 30 min before the ascorbic acid injection. This treatment was repeated again after 24 hr. They were sacrificed 48 hr later. Tyrosine hydroxylase was assayed in adrenal homogenates. Values represent the mean for three guinea pigs f SE. This group was fed a normal diet. FIG.
ON TYROSINE
519
HYDROXYLASE
immunochemical analysis. The specific activities in lOrj,OOOg supernatant fractions from the nonscorbutic and scorbutic guinea pigs were 3.81 nmoles per mg protein and 1.97 nmoles, respectively. For the experiment shown in Fig. 3, a constant amount of antiserum (0.5 ml) was added to increasing volumes of supernatant fluid of the nonscorbutic and scorbutic guinea pigs. The quantity of supernatant fluid of the nonscorbutic and scorbutic guinea pigs, which completely titrated the same amount of antibody, was 0.09 and 0.18 ml, respectively. ascorbic acid adminisTherefore, after tration, the amount of immunochemically reactive tyrosine hydroxylase increased 2-fold. Thus, the increase in tyrosine hydroxylase activity by the administration of 5.01
administration of ascorbic acid to the scorbutic guinea pigs stimulated protein synthesis involved in tyrosine hydroxylase activity. One possible explanation for these results was that the changes in tyrosine hydroxylase activity that followed ascorbic acid administration were due to changing amounts of enzyme protein. Therefore, we measured the levels of tyrosine hydroxylase protein in the adrenal gland of the guinea pig by immunochemical titration. Tyrosine Hytlroxylase Content of the Nonscorbutic and Scorbutic Guinea Pigs The scorbutic guinea pigs weighing about 300 g were separated into two groups. One group was ad.ministered 200 mg of ascorbic acid per day by intraperitoneal injection (the nonscorbutic group). The other group was given the same volume of 0.9 % NaCl solution (the scorbutic group). The adrenal glands of the nonscorbutic and scorbutic guinea pigs were homogenized and centrifuged for 60 min at 105,OOOg, and the resulting supernatant fluid was used for
SUPERNATANT
(ml)
3. The effect of administration of ascorbic acid on tyrosine hydroxylase content in the adrenal glands of guinea pigs. A constant amount of antiserum (0.5 ml) was added to increasing quantities of 105,000g supernatant solutions of adrenal gland prepared from the nonscorbutic (0) and scorbutic (X) guinea pigs. The tyrosine hydroxylase activity found after removal of the antigen-antibody precipitate is indicated in the ordinate. FIG.
520
NAKASHIMA
ascorbic acid was due to an increase in the amount of enzyme protein. These experiments indicate that the differences in specific activity of tyrosine hydroxylase seen upon ascorbic acid administration are actually due to the different amounts of enzyme in the adrenal glands of these animals. Thus, the change in tyrosine hydroxylase activity in the nonscorbutic and scorbutic guinea pigs can be entirely explained on the basis of changing amount of enzyme. DISCUSSION
In the present paper, it was demonstrated that tyrosine hydroxylase activity in adrenal gland of guinea pig decreased in scurvy, and it was recovered by the administration of ascorbic acid. This alteration might be due to the following causes. (a) Directly, ascorbic acid activated the enzyme. (b) Ascorbic acid affected the activation of low molecular weight coenzyme or metal ion such as Fe2+ (9). (c) Ascorbic acid increased the amount of protein such as cytochrome P-450 (10) involved in the enzyme activation. (d) Ascorbic acid changed the levels of tyrosine hydroxylase in adrenal gland of guinea pig. As indicated in a previous study (5), when 1 X lOW-1 X 1O-4 M ascorbic acid was added to the homogenate of the scorbutic guinea pig, the enzyme activity was not stimulated. Therefore, ascorbic acid did not activate the enzyme directly. Shiman et al. (11) demonstrated that tyrosine hydroxylase was inactivat,ed by Hz02 that was generated during the nonenzymatic oxidation of tetrahydropterin, and could be protected by catalase, peroxidase, or Fez+ from Hz02-mediated inactivation. Sufficient amounts of Fe2+ were added to the react,ion mixture in our experiment. Therefore, it was not considered that the catalase and peroxidase protected the enzyme activity of the nonscorbutic guinea pig and did not protect the enzyme activity of the scorbutic guinea pig from HzOzmediated inactivation. The experiments of gel filtration and enzyme purification were performed using 105,OOOg supernatant fraction. The data concerning the intracellular localization of tyrosine hydroxylase were rather conflicting.
ET AL.
Nagatsu et al. (9) and Pet,rack et al. (14) reported that tyrosine hydroxylase activity of bovine adrenal medulla was associated with particles. Stjarne and Lishajko (15), however, showed that this enzyme was found in a supernatant fraction. In this paper, when the adrenal glands were homogenized by a Potter-Elvehjem homogenizer, tyrosine hydroxylase activity was found in the 105,OOOgsupernatant’ fraction. Therefore, these experiments were performed using supernatant fraction. ACKNOWLEDGMENT The authors express their thanks Potjanee Threeratana (Ramathibody Bangkok, Thailand) for her excellent assistance.
to Miss Hospital, technical
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