Neurochemical Research (I) 337-347 (1976)
DOPAMINE-fl-HY DROXYLASE: S T I M U L A T I O N BY NITRO GEN-CONTAININ G HETEROCYCLICS AND THE ROLE OF C A T A L A S E 1 F. C. BROWN AND J. D . HARRALSON Tennessee Psychiatric Hospital and Institute and University of Tennessee Center for Health Sciences Memphis, Tennessee
A c c e p t e d March, 24, 1976
T h e catalase inhibitor 3-amino-l,2,4-triazole c a u s e s an increase in dopamine-,8h y d r o x y l a s e ( D B H ) activity, as do other nitrogen-containing heterocyclics, D e n a t u r e d catalase also c a u s e s an increase in activity, but in both cases, o p t i m u m activity is attained only in the p r e s e n c e of s o m e native catalase. It is proposed that the latter affects t h e D B H reaction in two different ways: It d e c o m p o s e s toxic peroxides, and it stabilizes the e n z y m e in s o m e m a n n e r as yet u n k n o w n , as do the heterocyclics. T h e nitrogen-containing c o m p o u n d s , and denatured catalase, protect D B H from inhibition by copper. Ideas concerning the relationships of copper, cataiase, and D B H m u s t be altered to a c c o m m o d a t e t h e s e n e w data.
INTRODUCTION In a recent article, we reported that the inhibition of dopamine-flhydroxylase [3,4-dihydroxyphenylethylamine, ascorbic: oxidoreductase (hydroxylating) E.C.1.14.2.1] (DBH) by copper was indirect, and suggested that the effect could be explained by an interaction of copper, 1 This study was supported in part by United States Public Health Service Grant NS04454. A preliminary report of t h e s e experiments was p r e s e n t e d at a S y m p o s i u m on N e w First and Second M e s s e n g e r s in N e r v o u s T i s s u e s in Brescia, Italy, A u g u s t 28-30, 1975.
337 9 1976 Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher.
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BROWN AND HARRALSON
ascorbic acid, and catalase (1). The latter is converted to the inactive catalase-H202(II) by the autoxidation of ascorbate and other electron donors. Copper accelerates the autoxidation, thereby enhancing the inactivation of catalase. D B H , which requires catalase, is therefore inhibited. The proposal was entirely consistent with prior knowledge of the p h e n o m e n a associated with both dopamine-/3-hydroxylase and catalase, but was not quite satisfying in one major respect. A nagging inconsistency was the question as to why such large quantities of catalase were needed to offset the effects of copper, particularly in the presence of tissue. In pursuit of an answer to this question, the experiments described in this paper were devised. The results suggest that the proposals offered by us, and by others (2,3), to explain the role of catalase in the D B H reaction must be modified and expanded.
EXPERIMENTAL
PROCEDURE
DBH was isolated and assayed by procedures that have been previously cited (1). In the current study, only one assay, a simple modification of the procedure described by Foldes et al. (4), was used. Each reaction vessel contained the following in a total volume of 0.25 ml: sodium acetate buffer, 30 ~mol (pH 5.0); ascorbic acid, 1.25 ~mol; fumaric acid, 12.5 ~mol; pargyline, 0.15 ~mol; (~H)-tyramine, 1 ~mol (specific activity 1 ixCi//xmol); and catalase, The amount of catalase and other agents used varied with the experiment. The reaction was started after a 10-min preincubation period by the addition of DBH. The amount of 3H-octopamineformed was oxidized and estimated as previously described (1). Blanks consisted of all ingredients except the test component, which was either replaced with water or, if it was on an enzyme, was denatured by boiling before adding. The following compounds were obtained from commercial sources and used without further purification: 3-aminotriazole, histamine'HCl, and histidine.HCl (Nutritional Biochemicals, Inc.); triazole, cataiase, hematin, and N-ethylmaleimide (Sigma); imidazole, pyrazole, and thiazole (Aldrich Chem.); benzoate and thymol (Fisher Scientific). All other materials were described previously (1).
RESULTS When 3-aminotriazole, a well-known inhibitor of catalase (5), was added to D B H assay reaction mixtures, the expected inhibition did not occur. N o t only was there no inhibition, but also D B H was stimulated in the absence of catalase. The stimulation was concentration-dependent, reaching a maximum at 0.015 M aminotriazole, after which some decrease occurred (Fig. 1). The stimulation by aminotriazole was significant, amounting to about 40% of the activity obtained with optimum concentrations of catalase.
339
DOPAMINE-3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
6
4
0
0
~
0.015 M
0.030
M
0.045
M
CONCENTRATION
FIG. 1. Stimulation of DBH activity by N-containing heterocyclics. The incubation mixture, which is described in the text, contained 6.5/zg DBH (specific activity 45.4/zmol octopamine formed/rag per h), but no catalase. Controls contained DBH that had been boiled for 10 rain. Test substances were: imidazole, O - - O ; pyrazole, ~ - - ~ ; and A - - A , triazole,
This stimulation is shown in Fig. 1 and Table I, along with data for several other heterocyclic nitrogen compounds. The most effective of these compounds were the unsubstituted 1,2,4-triazole, and pyrazole. Imidazole, histidine, and histamine were also active, but to a lesser extent. The stimulation by nitrogen-containing heterocyclics was similar to catalase (2,3), in that it did not occur unless the agent was added prior to the addition of the DBH. The heterocyclics had no effect on Km with either ascorbate or tyramine as substrate. Typical data are shown in Fig. 2, in which ascorbate was the variable substrate. In Fig. 3 (curves 3 and 4), data from a time-sequence study showed
340
BROWN AND HARRALSON
TABLE I STIMULATION OF DOPAMINE-fl-HYDROXYLASE BY NITROGEN-CONTAINING HETEROCYCLICSa
Additive None Reagent None Triazole Pyrazole 3-Aminotriazole Imidazole Histamine Histidine
Catalase b Percent of total activity
9.8 55.4 54.8 34.4 28.6 19.6 11.8
+- 1.5 -+ 3.7 +-- 4.0 + 1.3 -+ 5.0 _+ 2.4 -+ 2.0
38.6 97.2 95.6 68.0 84.8 43.0 28.4
+ 2.1 -+ 2.8 +- 2.9 -+~ 3.8 -+ 4.4 -+ 2.5 --- 4.3
a Assays were performed as indicated in the text. Each reaction vessel contained the test reagent at 0.015 M, which was the optimum concentration for the stimulatory effect. Each vessel also contained 6.5 ~g D B H (specific activity 45.4/zmol octopamine formed/ mg per h). Total activity was the activity observed with an optimum quantity of catalase (specific activity 36,000 U/rag, Sigma Chemical), which in these experiments was about 1,700 U. Values are expressed as the mean _+ SD; n = 5. Catalase, 100 U, was added.
that the initial rate of the DBH reaction was 10% less in the presence of triazole than it was with optimum concentrations of catalase. After about 10 rain, however, the rate with triazole decreased sharply, while the catalase-dependent reaction continued linearly. There is no evidence that the nitrogen-containing heterocyclics could stimulate the reaction by destroying H202 per se (5). In our hands, triazole had no effect on the absorbance of HzO2 (240 nm) at pH 7.0, nor did it affect the decomposition of the latter by catalase at pH 7.0. At pH 5.0, the absorbance of H202 increased with time, but there was no apparent effect of the azole compounds, although catalase caused a relatively slow decrease in the absorbance (Brown and Dittmann, unpublished data). The experiments described heretofore were initiated in an effort to understand the effects of copper on DBH. It was of interest to reexamine the copper toxicity in light of the new developments. These results are presented in Tables III and IV. Depending on the amount of copper and the concentration of the agent (Table IV), the presence of triazole and pyrazole or boiled catalase prevents the inhibition by copper
DOPAMINE-/3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
341
( T a b l e I I I ) . T h e a c t i v i t y o b s e r v e d in t h e p r e s e n c e o f l o w l e v e l s o f a c t i v e c a t a l a s e (100 U ) m a y also b e p r o t e c t e d b y t h e s e c o m p o u n d s . A t t h e c o n c e n t r a t i o n o f c o p p e r u s e d in t h e s e e x p e r i m e n t s ( 4 x 10 -6 M), d e n a t u r e d c a t a l a s e c o m p l e t e l y r e l i e v e d t h e i n h i b i t i o n . T h i s r e s u l t is c o n s i s t e n t with p r e v i o u s l y r e p o r t e d o b s e r v a t i o n s (1) t h a t high c o n c e n t r a t i o n o f n a t i v e c a t a l a s e w o u l d p r e v e n t th e i n h i b i t i o n o f D B H b y c o p p e r , b u t s h o w s that t h e p r e v e n t i o n is n o n e n z y m a t i c . T h e d a t a in T a b l e s I I I a n d I V s u g g e s t t h a t F e +§ b e h a v e s d i f f e r e n t l y f r o m s o m e o f t h e o t h e r c o m p o u n d s in t h e s y s t e m u n d e r d i s c u s s i o n . F o r i n s t a n c e , in t h e c o n c e n t r a t i o n s u s e d in t h e s e e x p e r i m e n t s ( T a b l e I V ) , it did n o t p r o t e c t D B H f r o m i n h i b i t i o n b y c o p p e r .
T A B L E II EFFECTS OF DENATURED CATALASE ON ACTIVATION OF DOPAMINE-/~I-IYDROXYLASEa Additive None Reagent Catalase None 1,700 U 1,700 U (heated) Precipitate Supernatant 4,500 U 4,500 U (heated) Precipitate Supernatant Other Hematin 5 x 10.5 M 5 x 10.6 M Ferrous sulfate 2 x 10-4 M 5 x 10-6 M
Catalase b Percent of total activity
9.80 _+1.5 99.5 -+ 1.0
38.6 -+ 2.1 100 -+ 0.0
24.5 -+ 1.7 9.5 -+ 0.6 100 -+ 0.0
97.8 -+ 2.6 36.5 -+ 1.7 100 _+ 0.0
48.5 _+ 1.7 9.0 -+ 0.8
100 _+ 0.0 37.0 -+ 1.2
8.5 _+ 0.6 9.5 _+ 1.3
41.3 -+ 1.5 37.5 _+ 3.3
74.8 -+ 2.4 16.5 -+ 1.7
81.8 +- 2.2 36.5 _+ 1.9
Assay conditions are described in the text and in footnote a to Table I. Catalase was heated at 100~ for 10 min prior to centrifugation. Aliquots of the supernatant and of the precipitate, which had been resuspended so as to maintain a constant volume, were added to reaction vessels. Values are expressed as the mean + so; n = 4. b Catalase, 100 U, was added.
342
BROWN AND HARRALSON
30~
25~
20~ O X
15~
10~
5 m
0
20
40
80
160
1/s FIG. 2. Kinetic analysis of the DBH reaction in the presence of triazole ( 0 - - 0 , K,, = 1.61• 10-2 raM) and catalase ~ I - - I , K m = 1.43x 10-2 mM). The substrate was ascorbate, which varied in concentration from 6.25 to 50 raM. Other conditions are described in the text and in the caption to Fig. 1.
DISCUSSION Several years ago, Margoliash et al. (5) showed that 3-amino-l,2,4triazole irreversibly inhibited catalase activity in the presence of a continuous supply of H202. The inhibition occurred with ascorbic acid and other autooxidizable substances as sources of H202, and was independent of pH in the range from 5.5 to 9.0. Catalase-H202 (I)
343
DOPAMINE-fl-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
donors, such as ethanol or formate, prevented the inhibition, which occurred in vitro and in vivo. In the DBH reaction, under conditions very similar to those described by Margoliash et al. (5), catalase is added to destroy any toxic H202 formed during the reaction. However, the triazole compound did not cause a reduction of activity. Instead of the expected inhibition, DBH was stimulated. This stimulation occurred whether or not catalase was present. Other heterocyclics, including thiazole and pyridine, were also active, but substances known to be free-radical scavengers, such as thymol, ethanol, and benzoate, had no effect (Brown and Harralson, unpublished data). In light of these observations and the data reported above, it is
e~
IE Q:
O I,;J 2:
X
;5 0 I0
I
10
20
1
30
MINUTES
FIG. 3. Time-sequence study of the DBH reaction with triazole and catalase. Assays were performed as indicated in the text and in the caption to Fig. 1. The following additions were made: ~ - - ~ , none; x--x, 100 U catalase; 0 - - 0 , triazole (0.015 M); 9 1 6 9 1,700 U catalase; A - - A , 100 U catalase plus triazole (0.015 M).
344
BROWN
AND
HARRALSON
TABLE
III
EFFECTS OF VARIOUS COMPOUNDS ON INHIBITION OF D O P A M I N E - ~ HYDROXYLASE BY COPPER a
Additives Catalase b Compound Azoles None Triazole
Pyrazole Imid azole
Histamine Histidine 3-Aminotriazo le Catalase
None 1,700 1,700 4,500 4,500
U U (heated) U U (heated)
Catalase b + Copper
Percent of total activity
38.6 97.2 95.6 84.8 43.0 28.4 68.0
-+ 444--44-
2.1 2.8 2.9 4.4 2.5 4.3 3.8
3.8 95.6 97.3 55.5 19.3 17.3 54.5
+ + 4444-
1.0 3.0 2.2 2.1 1.0 1.0 3.2
38.6 100 97.8 100 100
_+ 2.1 --- 0.0 + 2.6 4- 0.0 4- 0.0
1.30 68.0 42.5 100 98.8
4+ + -+ 4-
1.5 2.5 2.1 0.0 1.5
Other Hematin
37.5 +_ 3.31
4.00 4- 1.0
81.8 4- 2.2 36.5 4- 1.9
44.5 4- 2.1 9.3 4- 1.0
Ferrous sulfate 2 x 10-4 M 5 x 10-s M
a A s s a y and other procedural details are described in footnote a to Tables I and II. A dditions of copper were made to 4 x 10 -6 M. Values are expressed as the m e a n 4- SD; n = 4or5. b Catalase, 100 U, was added.
interesting to reexamine the role of catalase in the DBH reaction. The data in Fig. 1 and in Fig. 3 (curves 3 and 4) pose two interesting questions. Given the knowledge that the DBH reaction is inhibited unless it is initiated in the presence of catalase (2,3), presumably because of H202 toxicity, why does it proceed linearly for about 5-10 min in the presence of triazole alone. And why is the catalase not inhibited? Within the limits of current information, the most plausible explanation for the data in Fig. 3 is that catalase has two functions: (1) It decomposes peroxides that, in its absence, lead to the inhibition noted in
345
DOPAMINE-/3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
curve 3, Fig. 3; and (2) it has a stimulatory role analogous to, or identical to, that of the heterocyclic compounds. It has been known for some time that the amount of catalase required to give optimum DBH activity may vary (2,6), presumably again as needed for peroxide destruction, and may be inordinately high (1). Surprisingly, the data in Table I and Fig. 3 (curves 2 and 4) show that the amount of catalase required to give optimum activity can be lowered by at least 15-fold if appropriate concentrations of heterocyclics are incorporated into the reaction mixture. In other words, at low concentrations of catalase (100 U), the effects of the azole compounds appear to be additive to that of catalase. The possible exception in Table I is imidazole, which appears to stimulate more in the presence of catalase. However, maximum activity could never be obtained unless some catalase was present. These data support the hypothesis stated above that catalase has two functions and suggest the possibility that a portion of the added catalase, or something in the catalase preparation, is acting nonenzymatically. The data in Table II show this possibility to be the case. When the catalase preparation was boiled for 10 rain and centrifuged, approximately 35-50% of its stimulatory action was retained in the precipitate;
TABLE IV EFFECTS OF TRIAZOLE AND F e ++ ON INHIBITION OF DOPAMINE-~HYDROXYEASE BY COPPERa Copper (~M) 1
2
Reagent (raM) Triazole 7.5 15 30 Ferrous sulfate 0.1 0.2 0.5 1.0
5
10
Activity (%)
87 100 100
73 95 100
58 83 95
40 65 77
81 84 87 87
60 64 63 68
41 41 44 50
17 22 27 27
Assays were performed as indicated in the text and in footnote a to Table I. No catalase was added. Optimal DBH activity was obtained with 15 mM triazole and 0.2 mM Fe ++.
346
BROWN A N D H A R R A L S O N
the supernatant had no effect. When a small amount (190 U) of undenatured catalase was added, full DBH activity was restored (Table II). The nonenzymatic stimulation of DBH activity by catalase preparations is caused by a factor or factors as yet unknown. The data in Table II show that ferrous sulfate increases DBH activity as effectively as do the azole compounds. This property of Fe ++ was reported several years ago (3,6), but was considered to be, like the action of catalase, protection from inactivation by peroxides. Although the data in Table 2 do not rule out the possibility, they show that the catalase factor is probably not iron. The latter is associated with catalase in the form of hematin, which has no effect (Table II). The amount of iron in the quantities of catalase used in these experiments was calculated to be about 5 / , M , which is also ineffective (Table II). In all the experiments conducted to date, maximum activity is obtained only if some native catalase is present. In the absence of evidence to the contrary, it can be assumed that some catalase, or perhaps Fe ++, is needed to protect DBH from peroxides, as previously suggested (3). The denatured catalase, and the various heterocyclic compounds, appear to have a different type of stabilizing effect. The basis for this conjecture is that as much as 50% of the total DBH reaction can be obtained in the complete absence of catalase, but only if the stabilizing materials are in the reaction mixture prior to the addition of DBH. The "stabilizing" effect of denatured catalase has been previously reported (7). Although the possibility that these compounds may interact with peroxide in some way has not been ruled out, the data indicate that they do not protect DBH from peroxides as such. The effects of copper on the DBH reaction must also be reevaluated in view of the current data. Our earlier suggestion that copper inhibits the reaction through an indirect effect on catalase appears to be inadequate. Certainly, the reaction is not inhibited because catalase has been converted to catalase-H202 (II), as suggested. It now appears that the extra catalase and the azoles react directly with the copper to prevent the inhibition, either by destroying its capacity to stimulate peroxide production, or in some fashion as yet unknown. This tentative conclusion is based on the known property of the agents in question to form complexes with copper (8; Brown, unpublished observations). Also, the interaction is concentration-dependent, both in regard to copper and to the various agents, including catalase. There is as yet no evidence as to why catalase is not inhibited by triazole. However, the DBH reaction medium is a complex mixture, and it is possible that the interaction between triazole and catalase is
DOPAMINE-fl-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
347
p r e v e n t e d b y a d d i t i v e s such as the s u b s t r a t e , t y r a m i n e , or b y f u m a r a t e . T h e r e is also n o e v i d e n c e as to h o w a n y o f the o b s e r v e d r e a c t i o n s are m e d i a t e d . F u r t h e r m o r e , their s i g n i f i c a n c e to u n d e r s t a n d i n g the m e c h a n i s m s o f D B H a c t i v i t y , in v i t r o o r in v i v o , is u n k n o w n . E x p e r i m e n t s d e s i g n e d to a n s w e r t h e s e q u e s t i o n s a n d to e x p l o r e their p h y s i o l o g i c a l r e l e v a n c e are c u r r e n t l y in p r o g r e s s .
ACKNOWLEDGMENTS W e t h a n k A n n a D i t t m a n n for c o n t r i b u t i n g s o m e e x c e l l e n t t e c h n i c a l assistance.
REFERENCES 1. BROWN, F. C., and HARRALSON, J. D. (1975) Observations on the properties of dopamine-/3-hydroxylase. J. Neurochem. 24, 467-470. 2. LEVIN, E. Y., and KAUFMAN, S. (1961) Studies on the enzyme catalyzing the conversion of 3,4-dihydroxyphenylethylamine to norepinephrine. J. Biol. Chem. 236, 2043-2049. 3. GOLDSTEIN, M., LAUBER, E., and MCKEm~Gr~AN, M. R. (1965) Studies on the purification and characterization of 3,4-dehydroxypheny[ethylamine/3-hydroxylase. J. Biol. Chem. 240, 2066-2072. 4. FOLDES, A., JEFFREY, P. L., PRESTON, B. N., and AUSTIN, L. (1972) Dopamine-/3hydroxylase of bovine adrenal medulla. Biochem. J. 126, 120%1217. 5. MARGOLIASH,E., NOVOGRODSKY,A., and SCHEJXER, A. (1960) Irreversible reaction of 3-amino-l,2,4-triazole and related inhibitors with the protein of catalase. Biochem. J. 74, 339-348. 6. FRIEDMAN,S., and KAUFMAI%S. (1965) 3,4-Dihydroxyphenylethytamine-2,/3-hydroxylase. J. Biol. Chem. 240, 4763-4773. 7. AUNIS, D., M1RAs-PORTUGAL, M. T., and MANDEL, P. (1973) Bovine adrenal medullary dopamine-fl-hydroxylase: Purification by affinity chromatography, kinetic studies, and presence of essential histidyl residues. Biochim. Biophys. Acta 327, 313327. 8. HEMMER1CH,P. (1966) Model studies on the binding of univalent and redox-active copper in proteins, in PE~SACH, J., AISEN, P., and BLUMBER/3,W. F. (eds.); The Biochemistry of Copper, Academic Press, New York, pp. 15-34.
DOPAMINE-fl-HY DROXYLASE: S T I M U L A T I O N BY NITRO GEN-CONTAININ G HETEROCYCLICS AND THE ROLE OF C A T A L A S E 1 F. C. BROWN AND J. D . HARRALSON Tennessee Psychiatric Hospital and Institute and University of Tennessee Center for Health Sciences Memphis, Tennessee
A c c e p t e d March, 24, 1976
T h e catalase inhibitor 3-amino-l,2,4-triazole c a u s e s an increase in dopamine-,8h y d r o x y l a s e ( D B H ) activity, as do other nitrogen-containing heterocyclics, D e n a t u r e d catalase also c a u s e s an increase in activity, but in both cases, o p t i m u m activity is attained only in the p r e s e n c e of s o m e native catalase. It is proposed that the latter affects t h e D B H reaction in two different ways: It d e c o m p o s e s toxic peroxides, and it stabilizes the e n z y m e in s o m e m a n n e r as yet u n k n o w n , as do the heterocyclics. T h e nitrogen-containing c o m p o u n d s , and denatured catalase, protect D B H from inhibition by copper. Ideas concerning the relationships of copper, cataiase, and D B H m u s t be altered to a c c o m m o d a t e t h e s e n e w data.
INTRODUCTION In a recent article, we reported that the inhibition of dopamine-flhydroxylase [3,4-dihydroxyphenylethylamine, ascorbic: oxidoreductase (hydroxylating) E.C.1.14.2.1] (DBH) by copper was indirect, and suggested that the effect could be explained by an interaction of copper, 1 This study was supported in part by United States Public Health Service Grant NS04454. A preliminary report of t h e s e experiments was p r e s e n t e d at a S y m p o s i u m on N e w First and Second M e s s e n g e r s in N e r v o u s T i s s u e s in Brescia, Italy, A u g u s t 28-30, 1975.
337 9 1976 Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher.
338
BROWN AND HARRALSON
ascorbic acid, and catalase (1). The latter is converted to the inactive catalase-H202(II) by the autoxidation of ascorbate and other electron donors. Copper accelerates the autoxidation, thereby enhancing the inactivation of catalase. D B H , which requires catalase, is therefore inhibited. The proposal was entirely consistent with prior knowledge of the p h e n o m e n a associated with both dopamine-/3-hydroxylase and catalase, but was not quite satisfying in one major respect. A nagging inconsistency was the question as to why such large quantities of catalase were needed to offset the effects of copper, particularly in the presence of tissue. In pursuit of an answer to this question, the experiments described in this paper were devised. The results suggest that the proposals offered by us, and by others (2,3), to explain the role of catalase in the D B H reaction must be modified and expanded.
EXPERIMENTAL
PROCEDURE
DBH was isolated and assayed by procedures that have been previously cited (1). In the current study, only one assay, a simple modification of the procedure described by Foldes et al. (4), was used. Each reaction vessel contained the following in a total volume of 0.25 ml: sodium acetate buffer, 30 ~mol (pH 5.0); ascorbic acid, 1.25 ~mol; fumaric acid, 12.5 ~mol; pargyline, 0.15 ~mol; (~H)-tyramine, 1 ~mol (specific activity 1 ixCi//xmol); and catalase, The amount of catalase and other agents used varied with the experiment. The reaction was started after a 10-min preincubation period by the addition of DBH. The amount of 3H-octopamineformed was oxidized and estimated as previously described (1). Blanks consisted of all ingredients except the test component, which was either replaced with water or, if it was on an enzyme, was denatured by boiling before adding. The following compounds were obtained from commercial sources and used without further purification: 3-aminotriazole, histamine'HCl, and histidine.HCl (Nutritional Biochemicals, Inc.); triazole, cataiase, hematin, and N-ethylmaleimide (Sigma); imidazole, pyrazole, and thiazole (Aldrich Chem.); benzoate and thymol (Fisher Scientific). All other materials were described previously (1).
RESULTS When 3-aminotriazole, a well-known inhibitor of catalase (5), was added to D B H assay reaction mixtures, the expected inhibition did not occur. N o t only was there no inhibition, but also D B H was stimulated in the absence of catalase. The stimulation was concentration-dependent, reaching a maximum at 0.015 M aminotriazole, after which some decrease occurred (Fig. 1). The stimulation by aminotriazole was significant, amounting to about 40% of the activity obtained with optimum concentrations of catalase.
339
DOPAMINE-3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
6
4
0
0
~
0.015 M
0.030
M
0.045
M
CONCENTRATION
FIG. 1. Stimulation of DBH activity by N-containing heterocyclics. The incubation mixture, which is described in the text, contained 6.5/zg DBH (specific activity 45.4/zmol octopamine formed/rag per h), but no catalase. Controls contained DBH that had been boiled for 10 rain. Test substances were: imidazole, O - - O ; pyrazole, ~ - - ~ ; and A - - A , triazole,
This stimulation is shown in Fig. 1 and Table I, along with data for several other heterocyclic nitrogen compounds. The most effective of these compounds were the unsubstituted 1,2,4-triazole, and pyrazole. Imidazole, histidine, and histamine were also active, but to a lesser extent. The stimulation by nitrogen-containing heterocyclics was similar to catalase (2,3), in that it did not occur unless the agent was added prior to the addition of the DBH. The heterocyclics had no effect on Km with either ascorbate or tyramine as substrate. Typical data are shown in Fig. 2, in which ascorbate was the variable substrate. In Fig. 3 (curves 3 and 4), data from a time-sequence study showed
340
BROWN AND HARRALSON
TABLE I STIMULATION OF DOPAMINE-fl-HYDROXYLASE BY NITROGEN-CONTAINING HETEROCYCLICSa
Additive None Reagent None Triazole Pyrazole 3-Aminotriazole Imidazole Histamine Histidine
Catalase b Percent of total activity
9.8 55.4 54.8 34.4 28.6 19.6 11.8
+- 1.5 -+ 3.7 +-- 4.0 + 1.3 -+ 5.0 _+ 2.4 -+ 2.0
38.6 97.2 95.6 68.0 84.8 43.0 28.4
+ 2.1 -+ 2.8 +- 2.9 -+~ 3.8 -+ 4.4 -+ 2.5 --- 4.3
a Assays were performed as indicated in the text. Each reaction vessel contained the test reagent at 0.015 M, which was the optimum concentration for the stimulatory effect. Each vessel also contained 6.5 ~g D B H (specific activity 45.4/zmol octopamine formed/ mg per h). Total activity was the activity observed with an optimum quantity of catalase (specific activity 36,000 U/rag, Sigma Chemical), which in these experiments was about 1,700 U. Values are expressed as the mean _+ SD; n = 5. Catalase, 100 U, was added.
that the initial rate of the DBH reaction was 10% less in the presence of triazole than it was with optimum concentrations of catalase. After about 10 rain, however, the rate with triazole decreased sharply, while the catalase-dependent reaction continued linearly. There is no evidence that the nitrogen-containing heterocyclics could stimulate the reaction by destroying H202 per se (5). In our hands, triazole had no effect on the absorbance of HzO2 (240 nm) at pH 7.0, nor did it affect the decomposition of the latter by catalase at pH 7.0. At pH 5.0, the absorbance of H202 increased with time, but there was no apparent effect of the azole compounds, although catalase caused a relatively slow decrease in the absorbance (Brown and Dittmann, unpublished data). The experiments described heretofore were initiated in an effort to understand the effects of copper on DBH. It was of interest to reexamine the copper toxicity in light of the new developments. These results are presented in Tables III and IV. Depending on the amount of copper and the concentration of the agent (Table IV), the presence of triazole and pyrazole or boiled catalase prevents the inhibition by copper
DOPAMINE-/3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
341
( T a b l e I I I ) . T h e a c t i v i t y o b s e r v e d in t h e p r e s e n c e o f l o w l e v e l s o f a c t i v e c a t a l a s e (100 U ) m a y also b e p r o t e c t e d b y t h e s e c o m p o u n d s . A t t h e c o n c e n t r a t i o n o f c o p p e r u s e d in t h e s e e x p e r i m e n t s ( 4 x 10 -6 M), d e n a t u r e d c a t a l a s e c o m p l e t e l y r e l i e v e d t h e i n h i b i t i o n . T h i s r e s u l t is c o n s i s t e n t with p r e v i o u s l y r e p o r t e d o b s e r v a t i o n s (1) t h a t high c o n c e n t r a t i o n o f n a t i v e c a t a l a s e w o u l d p r e v e n t th e i n h i b i t i o n o f D B H b y c o p p e r , b u t s h o w s that t h e p r e v e n t i o n is n o n e n z y m a t i c . T h e d a t a in T a b l e s I I I a n d I V s u g g e s t t h a t F e +§ b e h a v e s d i f f e r e n t l y f r o m s o m e o f t h e o t h e r c o m p o u n d s in t h e s y s t e m u n d e r d i s c u s s i o n . F o r i n s t a n c e , in t h e c o n c e n t r a t i o n s u s e d in t h e s e e x p e r i m e n t s ( T a b l e I V ) , it did n o t p r o t e c t D B H f r o m i n h i b i t i o n b y c o p p e r .
T A B L E II EFFECTS OF DENATURED CATALASE ON ACTIVATION OF DOPAMINE-/~I-IYDROXYLASEa Additive None Reagent Catalase None 1,700 U 1,700 U (heated) Precipitate Supernatant 4,500 U 4,500 U (heated) Precipitate Supernatant Other Hematin 5 x 10.5 M 5 x 10.6 M Ferrous sulfate 2 x 10-4 M 5 x 10-6 M
Catalase b Percent of total activity
9.80 _+1.5 99.5 -+ 1.0
38.6 -+ 2.1 100 -+ 0.0
24.5 -+ 1.7 9.5 -+ 0.6 100 -+ 0.0
97.8 -+ 2.6 36.5 -+ 1.7 100 _+ 0.0
48.5 _+ 1.7 9.0 -+ 0.8
100 _+ 0.0 37.0 -+ 1.2
8.5 _+ 0.6 9.5 _+ 1.3
41.3 -+ 1.5 37.5 _+ 3.3
74.8 -+ 2.4 16.5 -+ 1.7
81.8 +- 2.2 36.5 _+ 1.9
Assay conditions are described in the text and in footnote a to Table I. Catalase was heated at 100~ for 10 min prior to centrifugation. Aliquots of the supernatant and of the precipitate, which had been resuspended so as to maintain a constant volume, were added to reaction vessels. Values are expressed as the mean + so; n = 4. b Catalase, 100 U, was added.
342
BROWN AND HARRALSON
30~
25~
20~ O X
15~
10~
5 m
0
20
40
80
160
1/s FIG. 2. Kinetic analysis of the DBH reaction in the presence of triazole ( 0 - - 0 , K,, = 1.61• 10-2 raM) and catalase ~ I - - I , K m = 1.43x 10-2 mM). The substrate was ascorbate, which varied in concentration from 6.25 to 50 raM. Other conditions are described in the text and in the caption to Fig. 1.
DISCUSSION Several years ago, Margoliash et al. (5) showed that 3-amino-l,2,4triazole irreversibly inhibited catalase activity in the presence of a continuous supply of H202. The inhibition occurred with ascorbic acid and other autooxidizable substances as sources of H202, and was independent of pH in the range from 5.5 to 9.0. Catalase-H202 (I)
343
DOPAMINE-fl-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
donors, such as ethanol or formate, prevented the inhibition, which occurred in vitro and in vivo. In the DBH reaction, under conditions very similar to those described by Margoliash et al. (5), catalase is added to destroy any toxic H202 formed during the reaction. However, the triazole compound did not cause a reduction of activity. Instead of the expected inhibition, DBH was stimulated. This stimulation occurred whether or not catalase was present. Other heterocyclics, including thiazole and pyridine, were also active, but substances known to be free-radical scavengers, such as thymol, ethanol, and benzoate, had no effect (Brown and Harralson, unpublished data). In light of these observations and the data reported above, it is
e~
IE Q:
O I,;J 2:
X
;5 0 I0
I
10
20
1
30
MINUTES
FIG. 3. Time-sequence study of the DBH reaction with triazole and catalase. Assays were performed as indicated in the text and in the caption to Fig. 1. The following additions were made: ~ - - ~ , none; x--x, 100 U catalase; 0 - - 0 , triazole (0.015 M); 9 1 6 9 1,700 U catalase; A - - A , 100 U catalase plus triazole (0.015 M).
344
BROWN
AND
HARRALSON
TABLE
III
EFFECTS OF VARIOUS COMPOUNDS ON INHIBITION OF D O P A M I N E - ~ HYDROXYLASE BY COPPER a
Additives Catalase b Compound Azoles None Triazole
Pyrazole Imid azole
Histamine Histidine 3-Aminotriazo le Catalase
None 1,700 1,700 4,500 4,500
U U (heated) U U (heated)
Catalase b + Copper
Percent of total activity
38.6 97.2 95.6 84.8 43.0 28.4 68.0
-+ 444--44-
2.1 2.8 2.9 4.4 2.5 4.3 3.8
3.8 95.6 97.3 55.5 19.3 17.3 54.5
+ + 4444-
1.0 3.0 2.2 2.1 1.0 1.0 3.2
38.6 100 97.8 100 100
_+ 2.1 --- 0.0 + 2.6 4- 0.0 4- 0.0
1.30 68.0 42.5 100 98.8
4+ + -+ 4-
1.5 2.5 2.1 0.0 1.5
Other Hematin
37.5 +_ 3.31
4.00 4- 1.0
81.8 4- 2.2 36.5 4- 1.9
44.5 4- 2.1 9.3 4- 1.0
Ferrous sulfate 2 x 10-4 M 5 x 10-s M
a A s s a y and other procedural details are described in footnote a to Tables I and II. A dditions of copper were made to 4 x 10 -6 M. Values are expressed as the m e a n 4- SD; n = 4or5. b Catalase, 100 U, was added.
interesting to reexamine the role of catalase in the DBH reaction. The data in Fig. 1 and in Fig. 3 (curves 3 and 4) pose two interesting questions. Given the knowledge that the DBH reaction is inhibited unless it is initiated in the presence of catalase (2,3), presumably because of H202 toxicity, why does it proceed linearly for about 5-10 min in the presence of triazole alone. And why is the catalase not inhibited? Within the limits of current information, the most plausible explanation for the data in Fig. 3 is that catalase has two functions: (1) It decomposes peroxides that, in its absence, lead to the inhibition noted in
345
DOPAMINE-/3-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
curve 3, Fig. 3; and (2) it has a stimulatory role analogous to, or identical to, that of the heterocyclic compounds. It has been known for some time that the amount of catalase required to give optimum DBH activity may vary (2,6), presumably again as needed for peroxide destruction, and may be inordinately high (1). Surprisingly, the data in Table I and Fig. 3 (curves 2 and 4) show that the amount of catalase required to give optimum activity can be lowered by at least 15-fold if appropriate concentrations of heterocyclics are incorporated into the reaction mixture. In other words, at low concentrations of catalase (100 U), the effects of the azole compounds appear to be additive to that of catalase. The possible exception in Table I is imidazole, which appears to stimulate more in the presence of catalase. However, maximum activity could never be obtained unless some catalase was present. These data support the hypothesis stated above that catalase has two functions and suggest the possibility that a portion of the added catalase, or something in the catalase preparation, is acting nonenzymatically. The data in Table II show this possibility to be the case. When the catalase preparation was boiled for 10 rain and centrifuged, approximately 35-50% of its stimulatory action was retained in the precipitate;
TABLE IV EFFECTS OF TRIAZOLE AND F e ++ ON INHIBITION OF DOPAMINE-~HYDROXYEASE BY COPPERa Copper (~M) 1
2
Reagent (raM) Triazole 7.5 15 30 Ferrous sulfate 0.1 0.2 0.5 1.0
5
10
Activity (%)
87 100 100
73 95 100
58 83 95
40 65 77
81 84 87 87
60 64 63 68
41 41 44 50
17 22 27 27
Assays were performed as indicated in the text and in footnote a to Table I. No catalase was added. Optimal DBH activity was obtained with 15 mM triazole and 0.2 mM Fe ++.
346
BROWN A N D H A R R A L S O N
the supernatant had no effect. When a small amount (190 U) of undenatured catalase was added, full DBH activity was restored (Table II). The nonenzymatic stimulation of DBH activity by catalase preparations is caused by a factor or factors as yet unknown. The data in Table II show that ferrous sulfate increases DBH activity as effectively as do the azole compounds. This property of Fe ++ was reported several years ago (3,6), but was considered to be, like the action of catalase, protection from inactivation by peroxides. Although the data in Table 2 do not rule out the possibility, they show that the catalase factor is probably not iron. The latter is associated with catalase in the form of hematin, which has no effect (Table II). The amount of iron in the quantities of catalase used in these experiments was calculated to be about 5 / , M , which is also ineffective (Table II). In all the experiments conducted to date, maximum activity is obtained only if some native catalase is present. In the absence of evidence to the contrary, it can be assumed that some catalase, or perhaps Fe ++, is needed to protect DBH from peroxides, as previously suggested (3). The denatured catalase, and the various heterocyclic compounds, appear to have a different type of stabilizing effect. The basis for this conjecture is that as much as 50% of the total DBH reaction can be obtained in the complete absence of catalase, but only if the stabilizing materials are in the reaction mixture prior to the addition of DBH. The "stabilizing" effect of denatured catalase has been previously reported (7). Although the possibility that these compounds may interact with peroxide in some way has not been ruled out, the data indicate that they do not protect DBH from peroxides as such. The effects of copper on the DBH reaction must also be reevaluated in view of the current data. Our earlier suggestion that copper inhibits the reaction through an indirect effect on catalase appears to be inadequate. Certainly, the reaction is not inhibited because catalase has been converted to catalase-H202 (II), as suggested. It now appears that the extra catalase and the azoles react directly with the copper to prevent the inhibition, either by destroying its capacity to stimulate peroxide production, or in some fashion as yet unknown. This tentative conclusion is based on the known property of the agents in question to form complexes with copper (8; Brown, unpublished observations). Also, the interaction is concentration-dependent, both in regard to copper and to the various agents, including catalase. There is as yet no evidence as to why catalase is not inhibited by triazole. However, the DBH reaction medium is a complex mixture, and it is possible that the interaction between triazole and catalase is
DOPAMINE-fl-HYDROXYLASE: EFFECTS OF AZOLES AND CATALASE
347
p r e v e n t e d b y a d d i t i v e s such as the s u b s t r a t e , t y r a m i n e , or b y f u m a r a t e . T h e r e is also n o e v i d e n c e as to h o w a n y o f the o b s e r v e d r e a c t i o n s are m e d i a t e d . F u r t h e r m o r e , their s i g n i f i c a n c e to u n d e r s t a n d i n g the m e c h a n i s m s o f D B H a c t i v i t y , in v i t r o o r in v i v o , is u n k n o w n . E x p e r i m e n t s d e s i g n e d to a n s w e r t h e s e q u e s t i o n s a n d to e x p l o r e their p h y s i o l o g i c a l r e l e v a n c e are c u r r e n t l y in p r o g r e s s .
ACKNOWLEDGMENTS W e t h a n k A n n a D i t t m a n n for c o n t r i b u t i n g s o m e e x c e l l e n t t e c h n i c a l assistance.
REFERENCES 1. BROWN, F. C., and HARRALSON, J. D. (1975) Observations on the properties of dopamine-/3-hydroxylase. J. Neurochem. 24, 467-470. 2. LEVIN, E. Y., and KAUFMAN, S. (1961) Studies on the enzyme catalyzing the conversion of 3,4-dihydroxyphenylethylamine to norepinephrine. J. Biol. Chem. 236, 2043-2049. 3. GOLDSTEIN, M., LAUBER, E., and MCKEm~Gr~AN, M. R. (1965) Studies on the purification and characterization of 3,4-dehydroxypheny[ethylamine/3-hydroxylase. J. Biol. Chem. 240, 2066-2072. 4. FOLDES, A., JEFFREY, P. L., PRESTON, B. N., and AUSTIN, L. (1972) Dopamine-/3hydroxylase of bovine adrenal medulla. Biochem. J. 126, 120%1217. 5. MARGOLIASH,E., NOVOGRODSKY,A., and SCHEJXER, A. (1960) Irreversible reaction of 3-amino-l,2,4-triazole and related inhibitors with the protein of catalase. Biochem. J. 74, 339-348. 6. FRIEDMAN,S., and KAUFMAI%S. (1965) 3,4-Dihydroxyphenylethytamine-2,/3-hydroxylase. J. Biol. Chem. 240, 4763-4773. 7. AUNIS, D., M1RAs-PORTUGAL, M. T., and MANDEL, P. (1973) Bovine adrenal medullary dopamine-fl-hydroxylase: Purification by affinity chromatography, kinetic studies, and presence of essential histidyl residues. Biochim. Biophys. Acta 327, 313327. 8. HEMMER1CH,P. (1966) Model studies on the binding of univalent and redox-active copper in proteins, in PE~SACH, J., AISEN, P., and BLUMBER/3,W. F. (eds.); The Biochemistry of Copper, Academic Press, New York, pp. 15-34.
