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The interactions between biogenic amines and pyridoxal, pyridoxal phosphate, and pyridoxal kinase
ARCHIVES
OF
BIOCHEMISTRY
AND
The Interactions Pyridoxal J. T. NEARY,2
BIO?HYSICS
161,
Between
4247 (1972)
Biogenic
Amines
and
Pyridoxal
Phosphate,
R. L. MENEELY,3
M. R. GREVER,
and
Pyridoxal,
Kinase’ AND
W. F. DIVEN
Departments of Biochemistry and Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania i&H5 Received January 26, 1972; accepted April 3, 1972 The effect of biogenic amines on pyridoxal kinase activity has been investigated in vitro. Experiments with a purified beef brain pyridoxal kinase indicate that 3,4dihydroxyphenylalanine (dopa), dopamine, norepinephrine, and serotonin inhibit the reaction, while tyrosine, tryptophan, and 5-hydroxytryptophan (5-HTP) have no effect. However, nonenzymatic studies under conditions similar to those of the enzymatic studies reveal that most of the inhibition by dopa, dopamine and norepinephrine is due to reaction of these compounds with pyridoxal phosphate while reaction between pyridoxal phosphate and the tryptophan derivatives, 5-HTP and serotonin, does not occur under the conditions employed. Additional nonenzymatic experiments indicate that the inhibition by serotonin is not due to removal of substrate (ATP, Zn2+, or pyridoxal). This evidence suggests that serotonin exerts its inhibitory effect by interacting with pyridoxal kinase. The enzyme inhibition observed with serotonin may represent a form of metabolic regulation.
In addition to being necessary for many general transamination, decarboxylation, and racemization reactions, pyridoxal phosphate is required as a coenzyme for metabolic reactions which are believed to be specifically related to the functional activity of the central nervous system (1). For example, pyridoxal phosphate-dependent decarboxylations lead to the formation of serotonin and dopamine, compounds that vary in amount for different behavioral states. The enzyme that catalyzes the production of pyridoxal phosphate from pyridoxal, ATP, and a divalent metal ion is pyridoxal kinase (ATP:
pyridoxal 5-phosphotransferase, EC 2.7. 1.35). From a series of in vivo experiments, Ebadi, Russell and McCoy (2) reported an inverse relationship between the activity of pyridoxal kinase in rabbit brain and the concentrations of brain norepinephrine, dopamine, and serotonin. The activity of the enzyme was low when the concentrations of the biogenic amines were elevated; conversely, the activity of the enzyme was high when the concentrations of the biogenic amines were decreased. Such findings suggest an interaction between the biogenic amines and pyridoxal kinase, but it is also conceivable that these compounds exert their influence by reacting directly with pyridoxal, thereby decreasing the substrate available for the enzyme, or by reacting with pyridoxal phosphate, thereby decreasing the amount of active cofactor available for metabolic reactions. For example, Hey1 et al. (3,4) and Schott and Clark (5) have shown that pyridoxal and pyridoxal phosphate can react with phenylalanine or phenethylamine de-
1 This research was supported in part by Grant L-18 from the Health Research Services Foundation. ) Traineeship recipient from United States Public Health Services Training Grant GM 00149. Present address, Thyroid Unit, Massachusetts General Hospital, Boston, MA 02114. a Recipient of a summer research fellowship from the Western Pennsylvania chapter of the Arthritis Foundation. 42 C opyright All
rights
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
BIOGENIC
AMINES
AND
rivatives that contain a meta-hydroxy substituent to form a tetrahydroisoquinoline condensation product. Studies with 3,4dihydroxyphen.ylalanine decarboxylase (5) and tyrosine transaminase (6,7) illustrate that this type of interaction can indeed lead to inhibition o:f enzymatic activity. With a high1.y purified preparation of beef brain pyridoxad kinase now available (8), it is possible to examine the effects of biogenic amines and their precursors on the pyridoxal kinase reaction. Here we present the results of in vitro enzymatic experiments and a nonenzymatic study of the interaction of the substrates anld product of the pyridoxal kinase reaction with some biogenic amines. MATERIALS
AND
METHODS
Chemicals. Pyridoxal, ATP (d&odium salt), pyridoxal phosphate, 3,4-dihydroxyphenylalanine (dopa) ,4 3-hydroxytyramine (dopamine), arterenol HCl (norepinephrine), 5hydroxytryptophan (5 HTP), 5-hydroxytyramine (serotonin, oxalate salt), tyrosine, and tryptophan were purchased from Sigma Chemical Corp. ZnCl? (analytical grade) was obtained from Mallinckrodt. Preparation fof beef brai,n pyridoxal kinase and measurements o.f enzymatic activity. Pyridoxal kinase was purified from beef brain as previously described (8). Enzymatic activity was determined by the calorimetric method of Wada and Snell (9), with the exception that the color development reaction was allowed to proceed for only 15 min, and by a spectrophotometric method (8). Interaction c,f substrates and product with biogenie amines. In the nonenzymatic studies, pyridoxal and pyridoxal phosphate concentrations were measured by the calorimetric methods of Wada and Sne.11 (9). Complex formation between biogenic amines and pyridoxal phosphate or pyridoxal was studied by recording absorption spectra on a Beckman Acta V spectrophotometer. Difference spectra for serotonin and substrate were obtained by recording the spectra of serotonin and substrate separately and the spectrum of a serotonin and substrate solution and then comparing the sum of the individual spectra to the spectrum of the serotonin and substrate solution. Zinc determination. The concentration of zinc in the presence and absence of serotonin was measured by the complexometric titration method as described by Schwarzenbach (10). 4 Abbreviations used: dopa, 3,4-dihydroxyphenylalanine; dopamine, 3-hydroxytyramine; Pal, pyridoxal; PALP, pyridoxal phosphate; 5-HTP, 5-hydroxytryptophan.
PYRIDOXAL
43
KINASE RESULTS
E$ect of biogenic amines on pyridoxal Cause reaction. The effect of biogenic amines on pyridoxal kinase activity was determined and the results with tyrosine and its catabolites from a typical series of experiments are presented in Table I. Tyrosine had no effect on the reaction, but apparent inhibition was observed in the presence of dopa, dopamine, and norepinephrine. The results of similar studies with tryptophan and its derivatives are shown in Table II. Apparent inhibition was observed only in the case of serotonin. Interaction between biogenic amines and pyridoxal and pyridoxal phosphate. Since interaction between pyridoxal and pyridoxal phosphate and biogenic amine-type compounds has been demonstrated (3-7)) a series of experiments were conducted to investigate the interaction between tyrosine and tryptophan derivatives and pyridoxal phosphate and pyridoxal under the conditions for enzymatic assay. The apparent inhibition of pyridoxal kinase suggested by the data presented in Tables I and II could have resulted from reaction of the biogenic amine compounds with pyridoxal phosphate thereby indicating a decrease in the amount produced during the time of the assay. Similarly, reaction could occur with pyridoxal thus reducTABLE I ACTIVITY OF PYRIDOX~L KINASE IN TIIE PRESENCE OF TYROSINE AND ITS DERIVATIVEV
Compound added
None Tyrosine Dopa Dopamine Norepinephrine
Pyridoxal phosphate formed (nmoles) 59 f 58 f 30 f 8fO 13 f
4 4 5 2
y0 Decrease of pyridoxal phosphate 0 0 49 86 78
a The incubation mixtures contained 1.6 mM 0.5 mM ATP, 0.16 mM Zn2+, 0.32 mM pyridoxal? tyrosine or derivative (where added), 110 rg protein, and 0.07 M KHzPO, buffer, pH 6.0. After a 30 min incubation at 37”C, the reaction was stopped and the amount of pyridoxal phosphate was determined. The results are given as the averages of duplicate determinations.
44
NEARY
ing the amount of substrate available to react with the enzyme. The concentration of free pyridoxal was measured in the presence and absence of the tyrosine or tryptophan derivatives after a 30 min incubation in 0.07 M KHzP04 buffer (pH 6.0) 37°C. As shown in Table III, no interaction was observed between pyridoxal and any of the compounds tested. The absorption spectra of 0.1 mM pyridoxal in phosphate buffer (pH 6.0) and mixtures of 0.1 mu pyridoxal and 0.1 mM tyrosine or tryptophan derivatives were determined from 250 to 500 nm in a BeckTABLE II ACTIVITY OF PYRIDOXAL KINASE IN THE PRESENCE OF TRYPTOPHAN AND ITS DERIVATIVES~
C;;$;Td
Pyridoxal phosphate formed (nmoles)
$$ Decrease of pyridoxal phosphate
55 f 4 55 f 4 54f4 5&O
0 0 0 91
None Tryptophan 5-HTP Serotonin
0 Pyridoxal kinase, 110 pg protein, was incubated with 1.6 mM ATP, 0.16 mM Zn2+, 0.32 mM pyridoxal, 0.5 mM tryptophan or derivative (where added) and 0.07 M KHzPO( buffer, pH 6.0. After 30 min at 37’, the reaction was stopped and the amount of pyridoxal phosphate was determined. The results are given as the averages of duplicate determinations.
ET AL.
man Acta V spectrophotometer. There was no evidence of complex formation. Similar studies were conducted with pyridoxal phosphate and the results of a typical series of experiments are presented in Table IV. Addition of dopa, dopamine, and norepinephrine resulted in decreased amounts of free pyridoxal phosphate while serotonin and 5-HTP had no effect. Spectrophotometric studies of complex formation between biogenic amines and pyridoxal phosphate. The results presented in Table IV suggest that complex formation occurs between pyridoxal phosphate and dopa, dopamine, and norepinephrine but not between pyridoxal phosphate and serotonin or 5HTP. However, it is possible that an indoleamine-pyridoxal phosphate complex is formed and then dissociated under the conditions employed to measure the amount of pyridoxal phosphate (calorimetric procedure of Wada and Snell). Thus, complex formation was measured directly by spectrophotometric methods. Studies of the absorption spectra of mixtures of biogenic amines and pyridoxal phosphate were consistent with the data shown in Table IV. The results of a typical study with dopa are presented in Fig. 1. Addition of increasing amounts of dopa to pyridoxal phosphate caused a decrease in the pyridoxal phosphate absorption TABLE IV EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOX~L PHOSPHATES
TABLE III EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOXAL~ Mixture Pal Pal Pal Pal Pal Pal
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
Pyridoxal 48 48 47 46 46 47
Mixture
Pyridoxal
Dxr!&e
of
phosphate pyfidoxal (nmoles)
phosphate
(nmoles) f f f f f f
1 0 1 0 0 1
o The reaction mixture contained 1.5 x 10e5 M pyridoxal, 5 X 10m4M tyrosine or tryptophan derivative (where added), and 0.07 M KHtPOl buffer, pH 6.0. After 30 min incubation at 37”C, the amount of pyridoxal was determined. The results are given as the averages of duplicate determinations.
0 32 34 56 5 6 ____ a A reaction mixture containing 2.5 X lop5 M pyridoxal phosphate, 5 X 10d4 M tyrosine or tryptophan derivative (where added), and 0.07 M KH~POP buffer (pH 6.0) was incubated for 30 min at 37°C. The free pyridoxal phosphate remaining at the end of the incubation was determined. The results are given as the averages of duplicate determinations. PALP PALP PALP PALP PALP PALP
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
84&l 56&O 54&O 37 f 1 80 f 1 79 f 4
BIOGENIC
AMINES
AND
PYRIDOXAL
45
KINASE TABLE
V
EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOX~L PHOSPHATE IN THE PRESENCE OF ATP, Zn++, AND PYRIDOX.~L~
Pyridoxd phosphate (nmoles)
Mixture
PALP PALP PALP PALP PALP PALP
WAVELENGTH
h-d
FIG. 1. Absorption spectra of 0.1 mM pyridoxal phosphate in 0.07 M phosphate buffer (pH 6.0) in the presence of varying concentrations of dopa. The mixtures of pyridoxal phosphate and dopa were incubated at 37°C for 30 min prior to determining the spectra. (1) pyridoxal phosphate alone; (2) 0.1 mM dopa; (3) 0.2 IIIM dopa; (4) 0.4 mM dopa; (5) 0.8 mM dopa; (6) dopa alone, 0.2 ml.
peak (388 nmj and the appearance of an absorption band with a peak at 325 nm. An isobestic point was observed at 345 nm. Black and Axelrod (6) presented similar spectrophotometric evidence for norepinephrine-pyridoxal phosphate interaction. In contrast to the catecholamine-pyridoxal phosphate experiments, similar studies of the spectra of pyridoxal phosphate in 0.07 M phosphate buffer (pH 6.0) with serotonin and 5-HTP gave no indication of complex formation. Thus it is not likely that the inhibition of pyridoxal kinase activity by serotonin (Table II) is due to the interaction of serotonin and pyridoxal phosphate. Substrate removal studies. As mentioned previously, complex formation between the biogenic amines and pyridoxal was not observed spectrophotometrically. The possibility of removal of ATP and Zn2f due to the presence of biogenic amines was also investigated. The results of an experiment in which biogenic amines were added to incubation solutions containing ATP, Zn2+, pyridoxal and pyridoxad phosphate in order to mimic the enzymatic assay conditions are shown in
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
81 51 59 46 77 82
f f f f f f
0 2 0 1 2 1
DecrrLe of pyridoxal phosphate 0 36 27 43 5 0
o A reaction mixture containing 2.5 X 10-s M pyridoxal phosphate, 5 X 10e4 M tyrosine or tryptophan derivative (where added), 1.6 mM ATP, 0.16 mM ZnZ+, 0.32 mM pyridoxal, 0.07 M KHZPO, buffer (pH 6.0) was incubated at 37°C for 30 min. The free pyridoxal phosphate was determined at the end of the incubation. The results are presented as the average of duplicate determinations.
Table V. The decreases in pyridoxal phosphate levels are similar to those obtained in the absence of ATP, Zn2+, and pyridoxal (Table IV), thereby indicating little if any interaction between ATP, zinc and the biogenie amines. Serotonin-ATP and serotonin-Zn interaction was examined by means of difference spectra. The spectra of 0.1 mM substrate, 0.1 mM serotonin, and 0.1 mM substrate plus 0.1 mM serotonin in 0.07 M KH2P04 buffer (pH 6.0) were recorded from 230 to 460 nm at room temperature on a Cary 14 spectrophotometer. The absorbance of the substrate plus serotonin mixture was compared to that of the sum of the substrate and serotonin. The lack of difference peaks for ATP plus serotonin and Zn plus serotonin indicate the absence of complex formation. A complexometric titration of 1 X 1O-3 M ZnClz in the presence and absence of serotonin (1 X 10-* M) gave the same amount of free Zn2+ for both cases. Under the conditions employed, no evidence has been found for complex formation between either ATP and serotonin or Zn2f and serotonin. DISCUSSION
The effect of catecholamines, indole amines, and their metabolic precursors on
46
NEARY
pyridoxal kinase activity has been investigated in vitro. Dopa, dopamine, norepinephrine, and serotonin appear to inhibit the enzyme, while 5HTP, tryptophan, and tyrosine have no effect on the activity (Tables I and II). However, nonenzymatic studies show that not all of the decrease in free pyridoxal phosphate production observed in the presence of the biogenic amines is due to direct interaction with the enzyme. None of the compounds studied appeared to interact significantly with pyridoxal under the conditions employed (Table III and absorbance spectra), thereby indicating that its removal by complex formation was not responsible for the apparent inhibition. However, dopa, dopamine, and norepinephrine did cause decreases in the levels of free pyridoxal phosphate (Tables IV and V and absorbance spectra). The difference in reactivity between pyridoxal and pyridoxal phosphate toward catecholamines can be explained by consideration of the equilibrium forms of pyridoxal and pyridoxal phosphate in aqueous solution. Near neutrality, pyridoxal is present almost entirely as the unreactive hemiacetal (1 l-13)) while pyridoxal phosphate, because of the phosphate group, exists in a free aldehyde form that can easily condense with catecholamines to give the tetrahydroisoquinoline derivatives. Although measurements of pyridoxal phosphate concentrations after incubation of dopa, dopamine, and norepinephrine with pyridoxa1 phosphate indicated complex formation, similar nonenzymatic experiments with serotonin and 5-HTP did not result in a change in the amount of pyridoxal phosphate. The possibility that the lack of decrease in pyridoxal phosphate in the presence of serotonin of 5-HTP is due to the formation of a reversible complex that dissociates upon the addition of acid (used to measure the level of pyridoxal phosphate according to the method of Wada and Snell) was considered unlikely since a decrease in pyridoxal phosphate was observed when serotonin was added to the pyridoxal kinase assay system (Table II). Both the enzymatic and nonenzymatic experiments employed the same method of pyridoxal phosphate measurement, so if addition of acid caused dissocia-
ET AL.
tion of a serotonin-pyridoxal phosphate complex, such a decrease would not have been observed. However, in order to study the interaction of serotonin and pyridoxal phosphate in a direct manner, absorption spectra were recorded. Addition of serotonin did not cause a change in the pyridoxal phosphate spectrum at the same pH, temperature, and time period employed in the enzymatic studies. Similar experiments with catecholamines (Fig. 1) did give a change in the absorption spectrum of pyridoxal phosphate, indicating complex formation. These findings are in accord with the chemical studies of Schott and Clark (5). The metahydroxy nature of the tyrosine derivatives is conducive to condensation with pyridoxal phosphate under the conditions employed, whereas the indole ring of the tryptophan derivatives is less favorable to a similar reaction. Thus, it appears that most of the “inhibition” of pyridoxal kinase by the tyrosine derivatives is due to interaction between the amines and pyridoxal phosphate, rather than a decrease in pyridoxal phosphate production. In a similar manner, inhibition of tyrosine transaminase by norepinephrine (6) and by dopa (7) has been attributed to the formation of a complex with pyridoxal phosphate. Interaction between indole amines and pyridoxal phosphate, however, has not been observed in these studies and it appears that inhibition of pyridoxal kinase by serotonin is the result of a direct interaction with the enzyme. In addition to the inhibition observed with the calorimetric assay for pyridoxal kinase activity, inhibition by serotonin has also been found by means of a spectrophotometric assay (8) for pyridoxal kinase activity that measures the initial rate of pyridoxal phosphate production directly (unpublished results). Thus, part of the in viva effect observed by Ebadi, Russell and McCoy (2) has been demonstrated in vitro, although no activation of pyridoxal kinase has been detected with the highly purified enzyme. The inhibition by serotonin may represent an interesting form of metabolir control. Tryptophan and 5-HTP do not decrease the activity of the kinase, thereby allowing the synthesis of pyridoxal phos-
BIOGENIC
AMINES
AND
phate which is required for the decarboxylation of 5-HTP to form serotonin. It is then possible that, when the in vivo concentration of serotonin reaches a significant level, pyridoxal kinase is inhibited and the synthesis of serotonin is prevented. When the concentration of serotonin decreases, pyridoxal phosphate formation can proceed and 5-HTP can be decarboxylated. This may be one way in which the levlel of a biogenic amine that is important in the functional activity of the brain is regulated. A different form of regulation has been postulated by Ebadi, Russell and McCoy (2). They suggest that the level of pyridoxal phosphate controls the activity of pyridoxal kinase, and that a change in the activity of pyridoxal kinase induced by the levels of the amines may be only secondary to the effects primarily induced by the presence or absence of pyridoxal phosphate. However, the direct interaction of serotonin with pyridoxal kinase, as described here, suggests that serotonin plays a stronger role in the regulation of pyridoxal k:inase than has been implicated by previous studies. Further studies to elucidate the mechanism by which serotonin inhibits pyridoxal kinase are now in progress.
PYRIDOXAL
KINASE
47
REFERENCES
5. 6. 7. 8. 9. 10.
11.
12. 13.
HOLTZ, P., AND P.~LM, D. (1964) Pharmacol. Rev. 16, 115. EBADI, M. S., RUSSELL, R. L., AND MCCOY, E. E. (1968) J. Neurochem. 16,659. HEYL, D., HARRIS, S. A., AND FOLKERS, K. (1948) J. Amer. Chem. Sot. 70,3669. HEYL, D., Luz, E., HARRIS, S. A., AND FOLKERS, K. (1952) J. Amer. Chem. Sot. 74, 414. SCHOTT, H. F., AND CLARK, W. G. (1952) J. Biol. Chem. 196, 449. BLACK, I. B., AND AXELROD, J. (1969) J. Biol. Chem. 244, 6124. FELLMAN, J. H., AND ROTH, E. S. (1971) Biochemistry 10, 408. NEARY, J. T., AND DIVEN, W. F. (1970) J. Biol. Chem. 246, 5585. WADA, H., AND SNELL, E. E. (1961) J. Biol. Chem. 236, 2089. SCHWARZENBACH, G. (1957) Complexometric Titrations, p. 83, Wiley (Interscience), New York. HEYL, D., Luz, E., HARRIS, S. A., AND FOLKERS, K. (1951) J. Amer. Chem. Sot. 73, 3430. METZLER, D. E., AND SNELL, E. E. (1955) J. Amer. Chem. Sot. ‘77, 2431. SNELL, E. E. (1958) Vitam. Horm. (New York) 16, 77.
OF
BIOCHEMISTRY
AND
The Interactions Pyridoxal J. T. NEARY,2
BIO?HYSICS
161,
Between
4247 (1972)
Biogenic
Amines
and
Pyridoxal
Phosphate,
R. L. MENEELY,3
M. R. GREVER,
and
Pyridoxal,
Kinase’ AND
W. F. DIVEN
Departments of Biochemistry and Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania i&H5 Received January 26, 1972; accepted April 3, 1972 The effect of biogenic amines on pyridoxal kinase activity has been investigated in vitro. Experiments with a purified beef brain pyridoxal kinase indicate that 3,4dihydroxyphenylalanine (dopa), dopamine, norepinephrine, and serotonin inhibit the reaction, while tyrosine, tryptophan, and 5-hydroxytryptophan (5-HTP) have no effect. However, nonenzymatic studies under conditions similar to those of the enzymatic studies reveal that most of the inhibition by dopa, dopamine and norepinephrine is due to reaction of these compounds with pyridoxal phosphate while reaction between pyridoxal phosphate and the tryptophan derivatives, 5-HTP and serotonin, does not occur under the conditions employed. Additional nonenzymatic experiments indicate that the inhibition by serotonin is not due to removal of substrate (ATP, Zn2+, or pyridoxal). This evidence suggests that serotonin exerts its inhibitory effect by interacting with pyridoxal kinase. The enzyme inhibition observed with serotonin may represent a form of metabolic regulation.
In addition to being necessary for many general transamination, decarboxylation, and racemization reactions, pyridoxal phosphate is required as a coenzyme for metabolic reactions which are believed to be specifically related to the functional activity of the central nervous system (1). For example, pyridoxal phosphate-dependent decarboxylations lead to the formation of serotonin and dopamine, compounds that vary in amount for different behavioral states. The enzyme that catalyzes the production of pyridoxal phosphate from pyridoxal, ATP, and a divalent metal ion is pyridoxal kinase (ATP:
pyridoxal 5-phosphotransferase, EC 2.7. 1.35). From a series of in vivo experiments, Ebadi, Russell and McCoy (2) reported an inverse relationship between the activity of pyridoxal kinase in rabbit brain and the concentrations of brain norepinephrine, dopamine, and serotonin. The activity of the enzyme was low when the concentrations of the biogenic amines were elevated; conversely, the activity of the enzyme was high when the concentrations of the biogenic amines were decreased. Such findings suggest an interaction between the biogenic amines and pyridoxal kinase, but it is also conceivable that these compounds exert their influence by reacting directly with pyridoxal, thereby decreasing the substrate available for the enzyme, or by reacting with pyridoxal phosphate, thereby decreasing the amount of active cofactor available for metabolic reactions. For example, Hey1 et al. (3,4) and Schott and Clark (5) have shown that pyridoxal and pyridoxal phosphate can react with phenylalanine or phenethylamine de-
1 This research was supported in part by Grant L-18 from the Health Research Services Foundation. ) Traineeship recipient from United States Public Health Services Training Grant GM 00149. Present address, Thyroid Unit, Massachusetts General Hospital, Boston, MA 02114. a Recipient of a summer research fellowship from the Western Pennsylvania chapter of the Arthritis Foundation. 42 C opyright All
rights
@ 1972 by Academic Press, of reproduction in any form
Inc. reserved.
BIOGENIC
AMINES
AND
rivatives that contain a meta-hydroxy substituent to form a tetrahydroisoquinoline condensation product. Studies with 3,4dihydroxyphen.ylalanine decarboxylase (5) and tyrosine transaminase (6,7) illustrate that this type of interaction can indeed lead to inhibition o:f enzymatic activity. With a high1.y purified preparation of beef brain pyridoxad kinase now available (8), it is possible to examine the effects of biogenic amines and their precursors on the pyridoxal kinase reaction. Here we present the results of in vitro enzymatic experiments and a nonenzymatic study of the interaction of the substrates anld product of the pyridoxal kinase reaction with some biogenic amines. MATERIALS
AND
METHODS
Chemicals. Pyridoxal, ATP (d&odium salt), pyridoxal phosphate, 3,4-dihydroxyphenylalanine (dopa) ,4 3-hydroxytyramine (dopamine), arterenol HCl (norepinephrine), 5hydroxytryptophan (5 HTP), 5-hydroxytyramine (serotonin, oxalate salt), tyrosine, and tryptophan were purchased from Sigma Chemical Corp. ZnCl? (analytical grade) was obtained from Mallinckrodt. Preparation fof beef brai,n pyridoxal kinase and measurements o.f enzymatic activity. Pyridoxal kinase was purified from beef brain as previously described (8). Enzymatic activity was determined by the calorimetric method of Wada and Snell (9), with the exception that the color development reaction was allowed to proceed for only 15 min, and by a spectrophotometric method (8). Interaction c,f substrates and product with biogenie amines. In the nonenzymatic studies, pyridoxal and pyridoxal phosphate concentrations were measured by the calorimetric methods of Wada and Sne.11 (9). Complex formation between biogenic amines and pyridoxal phosphate or pyridoxal was studied by recording absorption spectra on a Beckman Acta V spectrophotometer. Difference spectra for serotonin and substrate were obtained by recording the spectra of serotonin and substrate separately and the spectrum of a serotonin and substrate solution and then comparing the sum of the individual spectra to the spectrum of the serotonin and substrate solution. Zinc determination. The concentration of zinc in the presence and absence of serotonin was measured by the complexometric titration method as described by Schwarzenbach (10). 4 Abbreviations used: dopa, 3,4-dihydroxyphenylalanine; dopamine, 3-hydroxytyramine; Pal, pyridoxal; PALP, pyridoxal phosphate; 5-HTP, 5-hydroxytryptophan.
PYRIDOXAL
43
KINASE RESULTS
E$ect of biogenic amines on pyridoxal Cause reaction. The effect of biogenic amines on pyridoxal kinase activity was determined and the results with tyrosine and its catabolites from a typical series of experiments are presented in Table I. Tyrosine had no effect on the reaction, but apparent inhibition was observed in the presence of dopa, dopamine, and norepinephrine. The results of similar studies with tryptophan and its derivatives are shown in Table II. Apparent inhibition was observed only in the case of serotonin. Interaction between biogenic amines and pyridoxal and pyridoxal phosphate. Since interaction between pyridoxal and pyridoxal phosphate and biogenic amine-type compounds has been demonstrated (3-7)) a series of experiments were conducted to investigate the interaction between tyrosine and tryptophan derivatives and pyridoxal phosphate and pyridoxal under the conditions for enzymatic assay. The apparent inhibition of pyridoxal kinase suggested by the data presented in Tables I and II could have resulted from reaction of the biogenic amine compounds with pyridoxal phosphate thereby indicating a decrease in the amount produced during the time of the assay. Similarly, reaction could occur with pyridoxal thus reducTABLE I ACTIVITY OF PYRIDOX~L KINASE IN TIIE PRESENCE OF TYROSINE AND ITS DERIVATIVEV
Compound added
None Tyrosine Dopa Dopamine Norepinephrine
Pyridoxal phosphate formed (nmoles) 59 f 58 f 30 f 8fO 13 f
4 4 5 2
y0 Decrease of pyridoxal phosphate 0 0 49 86 78
a The incubation mixtures contained 1.6 mM 0.5 mM ATP, 0.16 mM Zn2+, 0.32 mM pyridoxal? tyrosine or derivative (where added), 110 rg protein, and 0.07 M KHzPO, buffer, pH 6.0. After a 30 min incubation at 37”C, the reaction was stopped and the amount of pyridoxal phosphate was determined. The results are given as the averages of duplicate determinations.
44
NEARY
ing the amount of substrate available to react with the enzyme. The concentration of free pyridoxal was measured in the presence and absence of the tyrosine or tryptophan derivatives after a 30 min incubation in 0.07 M KHzP04 buffer (pH 6.0) 37°C. As shown in Table III, no interaction was observed between pyridoxal and any of the compounds tested. The absorption spectra of 0.1 mM pyridoxal in phosphate buffer (pH 6.0) and mixtures of 0.1 mu pyridoxal and 0.1 mM tyrosine or tryptophan derivatives were determined from 250 to 500 nm in a BeckTABLE II ACTIVITY OF PYRIDOXAL KINASE IN THE PRESENCE OF TRYPTOPHAN AND ITS DERIVATIVES~
C;;$;Td
Pyridoxal phosphate formed (nmoles)
$$ Decrease of pyridoxal phosphate
55 f 4 55 f 4 54f4 5&O
0 0 0 91
None Tryptophan 5-HTP Serotonin
0 Pyridoxal kinase, 110 pg protein, was incubated with 1.6 mM ATP, 0.16 mM Zn2+, 0.32 mM pyridoxal, 0.5 mM tryptophan or derivative (where added) and 0.07 M KHzPO( buffer, pH 6.0. After 30 min at 37’, the reaction was stopped and the amount of pyridoxal phosphate was determined. The results are given as the averages of duplicate determinations.
ET AL.
man Acta V spectrophotometer. There was no evidence of complex formation. Similar studies were conducted with pyridoxal phosphate and the results of a typical series of experiments are presented in Table IV. Addition of dopa, dopamine, and norepinephrine resulted in decreased amounts of free pyridoxal phosphate while serotonin and 5-HTP had no effect. Spectrophotometric studies of complex formation between biogenic amines and pyridoxal phosphate. The results presented in Table IV suggest that complex formation occurs between pyridoxal phosphate and dopa, dopamine, and norepinephrine but not between pyridoxal phosphate and serotonin or 5HTP. However, it is possible that an indoleamine-pyridoxal phosphate complex is formed and then dissociated under the conditions employed to measure the amount of pyridoxal phosphate (calorimetric procedure of Wada and Snell). Thus, complex formation was measured directly by spectrophotometric methods. Studies of the absorption spectra of mixtures of biogenic amines and pyridoxal phosphate were consistent with the data shown in Table IV. The results of a typical study with dopa are presented in Fig. 1. Addition of increasing amounts of dopa to pyridoxal phosphate caused a decrease in the pyridoxal phosphate absorption TABLE IV EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOX~L PHOSPHATES
TABLE III EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOXAL~ Mixture Pal Pal Pal Pal Pal Pal
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
Pyridoxal 48 48 47 46 46 47
Mixture
Pyridoxal
Dxr!&e
of
phosphate pyfidoxal (nmoles)
phosphate
(nmoles) f f f f f f
1 0 1 0 0 1
o The reaction mixture contained 1.5 x 10e5 M pyridoxal, 5 X 10m4M tyrosine or tryptophan derivative (where added), and 0.07 M KHtPOl buffer, pH 6.0. After 30 min incubation at 37”C, the amount of pyridoxal was determined. The results are given as the averages of duplicate determinations.
0 32 34 56 5 6 ____ a A reaction mixture containing 2.5 X lop5 M pyridoxal phosphate, 5 X 10d4 M tyrosine or tryptophan derivative (where added), and 0.07 M KH~POP buffer (pH 6.0) was incubated for 30 min at 37°C. The free pyridoxal phosphate remaining at the end of the incubation was determined. The results are given as the averages of duplicate determinations. PALP PALP PALP PALP PALP PALP
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
84&l 56&O 54&O 37 f 1 80 f 1 79 f 4
BIOGENIC
AMINES
AND
PYRIDOXAL
45
KINASE TABLE
V
EFFECT OF BIOGENIC AMINES AND THEIR PRECURSORS ON PYRIDOX~L PHOSPHATE IN THE PRESENCE OF ATP, Zn++, AND PYRIDOX.~L~
Pyridoxd phosphate (nmoles)
Mixture
PALP PALP PALP PALP PALP PALP
WAVELENGTH
h-d
FIG. 1. Absorption spectra of 0.1 mM pyridoxal phosphate in 0.07 M phosphate buffer (pH 6.0) in the presence of varying concentrations of dopa. The mixtures of pyridoxal phosphate and dopa were incubated at 37°C for 30 min prior to determining the spectra. (1) pyridoxal phosphate alone; (2) 0.1 mM dopa; (3) 0.2 IIIM dopa; (4) 0.4 mM dopa; (5) 0.8 mM dopa; (6) dopa alone, 0.2 ml.
peak (388 nmj and the appearance of an absorption band with a peak at 325 nm. An isobestic point was observed at 345 nm. Black and Axelrod (6) presented similar spectrophotometric evidence for norepinephrine-pyridoxal phosphate interaction. In contrast to the catecholamine-pyridoxal phosphate experiments, similar studies of the spectra of pyridoxal phosphate in 0.07 M phosphate buffer (pH 6.0) with serotonin and 5-HTP gave no indication of complex formation. Thus it is not likely that the inhibition of pyridoxal kinase activity by serotonin (Table II) is due to the interaction of serotonin and pyridoxal phosphate. Substrate removal studies. As mentioned previously, complex formation between the biogenic amines and pyridoxal was not observed spectrophotometrically. The possibility of removal of ATP and Zn2f due to the presence of biogenic amines was also investigated. The results of an experiment in which biogenic amines were added to incubation solutions containing ATP, Zn2+, pyridoxal and pyridoxad phosphate in order to mimic the enzymatic assay conditions are shown in
+ + + + +
dopa dopamine norepinephrine 5-HTP serotonin
81 51 59 46 77 82
f f f f f f
0 2 0 1 2 1
DecrrLe of pyridoxal phosphate 0 36 27 43 5 0
o A reaction mixture containing 2.5 X 10-s M pyridoxal phosphate, 5 X 10e4 M tyrosine or tryptophan derivative (where added), 1.6 mM ATP, 0.16 mM ZnZ+, 0.32 mM pyridoxal, 0.07 M KHZPO, buffer (pH 6.0) was incubated at 37°C for 30 min. The free pyridoxal phosphate was determined at the end of the incubation. The results are presented as the average of duplicate determinations.
Table V. The decreases in pyridoxal phosphate levels are similar to those obtained in the absence of ATP, Zn2+, and pyridoxal (Table IV), thereby indicating little if any interaction between ATP, zinc and the biogenie amines. Serotonin-ATP and serotonin-Zn interaction was examined by means of difference spectra. The spectra of 0.1 mM substrate, 0.1 mM serotonin, and 0.1 mM substrate plus 0.1 mM serotonin in 0.07 M KH2P04 buffer (pH 6.0) were recorded from 230 to 460 nm at room temperature on a Cary 14 spectrophotometer. The absorbance of the substrate plus serotonin mixture was compared to that of the sum of the substrate and serotonin. The lack of difference peaks for ATP plus serotonin and Zn plus serotonin indicate the absence of complex formation. A complexometric titration of 1 X 1O-3 M ZnClz in the presence and absence of serotonin (1 X 10-* M) gave the same amount of free Zn2+ for both cases. Under the conditions employed, no evidence has been found for complex formation between either ATP and serotonin or Zn2f and serotonin. DISCUSSION
The effect of catecholamines, indole amines, and their metabolic precursors on
46
NEARY
pyridoxal kinase activity has been investigated in vitro. Dopa, dopamine, norepinephrine, and serotonin appear to inhibit the enzyme, while 5HTP, tryptophan, and tyrosine have no effect on the activity (Tables I and II). However, nonenzymatic studies show that not all of the decrease in free pyridoxal phosphate production observed in the presence of the biogenic amines is due to direct interaction with the enzyme. None of the compounds studied appeared to interact significantly with pyridoxal under the conditions employed (Table III and absorbance spectra), thereby indicating that its removal by complex formation was not responsible for the apparent inhibition. However, dopa, dopamine, and norepinephrine did cause decreases in the levels of free pyridoxal phosphate (Tables IV and V and absorbance spectra). The difference in reactivity between pyridoxal and pyridoxal phosphate toward catecholamines can be explained by consideration of the equilibrium forms of pyridoxal and pyridoxal phosphate in aqueous solution. Near neutrality, pyridoxal is present almost entirely as the unreactive hemiacetal (1 l-13)) while pyridoxal phosphate, because of the phosphate group, exists in a free aldehyde form that can easily condense with catecholamines to give the tetrahydroisoquinoline derivatives. Although measurements of pyridoxal phosphate concentrations after incubation of dopa, dopamine, and norepinephrine with pyridoxa1 phosphate indicated complex formation, similar nonenzymatic experiments with serotonin and 5-HTP did not result in a change in the amount of pyridoxal phosphate. The possibility that the lack of decrease in pyridoxal phosphate in the presence of serotonin of 5-HTP is due to the formation of a reversible complex that dissociates upon the addition of acid (used to measure the level of pyridoxal phosphate according to the method of Wada and Snell) was considered unlikely since a decrease in pyridoxal phosphate was observed when serotonin was added to the pyridoxal kinase assay system (Table II). Both the enzymatic and nonenzymatic experiments employed the same method of pyridoxal phosphate measurement, so if addition of acid caused dissocia-
ET AL.
tion of a serotonin-pyridoxal phosphate complex, such a decrease would not have been observed. However, in order to study the interaction of serotonin and pyridoxal phosphate in a direct manner, absorption spectra were recorded. Addition of serotonin did not cause a change in the pyridoxal phosphate spectrum at the same pH, temperature, and time period employed in the enzymatic studies. Similar experiments with catecholamines (Fig. 1) did give a change in the absorption spectrum of pyridoxal phosphate, indicating complex formation. These findings are in accord with the chemical studies of Schott and Clark (5). The metahydroxy nature of the tyrosine derivatives is conducive to condensation with pyridoxal phosphate under the conditions employed, whereas the indole ring of the tryptophan derivatives is less favorable to a similar reaction. Thus, it appears that most of the “inhibition” of pyridoxal kinase by the tyrosine derivatives is due to interaction between the amines and pyridoxal phosphate, rather than a decrease in pyridoxal phosphate production. In a similar manner, inhibition of tyrosine transaminase by norepinephrine (6) and by dopa (7) has been attributed to the formation of a complex with pyridoxal phosphate. Interaction between indole amines and pyridoxal phosphate, however, has not been observed in these studies and it appears that inhibition of pyridoxal kinase by serotonin is the result of a direct interaction with the enzyme. In addition to the inhibition observed with the calorimetric assay for pyridoxal kinase activity, inhibition by serotonin has also been found by means of a spectrophotometric assay (8) for pyridoxal kinase activity that measures the initial rate of pyridoxal phosphate production directly (unpublished results). Thus, part of the in viva effect observed by Ebadi, Russell and McCoy (2) has been demonstrated in vitro, although no activation of pyridoxal kinase has been detected with the highly purified enzyme. The inhibition by serotonin may represent an interesting form of metabolir control. Tryptophan and 5-HTP do not decrease the activity of the kinase, thereby allowing the synthesis of pyridoxal phos-
BIOGENIC
AMINES
AND
phate which is required for the decarboxylation of 5-HTP to form serotonin. It is then possible that, when the in vivo concentration of serotonin reaches a significant level, pyridoxal kinase is inhibited and the synthesis of serotonin is prevented. When the concentration of serotonin decreases, pyridoxal phosphate formation can proceed and 5-HTP can be decarboxylated. This may be one way in which the levlel of a biogenic amine that is important in the functional activity of the brain is regulated. A different form of regulation has been postulated by Ebadi, Russell and McCoy (2). They suggest that the level of pyridoxal phosphate controls the activity of pyridoxal kinase, and that a change in the activity of pyridoxal kinase induced by the levels of the amines may be only secondary to the effects primarily induced by the presence or absence of pyridoxal phosphate. However, the direct interaction of serotonin with pyridoxal kinase, as described here, suggests that serotonin plays a stronger role in the regulation of pyridoxal k:inase than has been implicated by previous studies. Further studies to elucidate the mechanism by which serotonin inhibits pyridoxal kinase are now in progress.
PYRIDOXAL
KINASE
47
REFERENCES
5. 6. 7. 8. 9. 10.
11.
12. 13.
HOLTZ, P., AND P.~LM, D. (1964) Pharmacol. Rev. 16, 115. EBADI, M. S., RUSSELL, R. L., AND MCCOY, E. E. (1968) J. Neurochem. 16,659. HEYL, D., HARRIS, S. A., AND FOLKERS, K. (1948) J. Amer. Chem. Sot. 70,3669. HEYL, D., Luz, E., HARRIS, S. A., AND FOLKERS, K. (1952) J. Amer. Chem. Sot. 74, 414. SCHOTT, H. F., AND CLARK, W. G. (1952) J. Biol. Chem. 196, 449. BLACK, I. B., AND AXELROD, J. (1969) J. Biol. Chem. 244, 6124. FELLMAN, J. H., AND ROTH, E. S. (1971) Biochemistry 10, 408. NEARY, J. T., AND DIVEN, W. F. (1970) J. Biol. Chem. 246, 5585. WADA, H., AND SNELL, E. E. (1961) J. Biol. Chem. 236, 2089. SCHWARZENBACH, G. (1957) Complexometric Titrations, p. 83, Wiley (Interscience), New York. HEYL, D., Luz, E., HARRIS, S. A., AND FOLKERS, K. (1951) J. Amer. Chem. Sot. 73, 3430. METZLER, D. E., AND SNELL, E. E. (1955) J. Amer. Chem. Sot. ‘77, 2431. SNELL, E. E. (1958) Vitam. Horm. (New York) 16, 77.