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Inhibition of phenylalanine hydroxylase activity by α-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase
Life Sciences Vol. 17, pp . Printed in the U.S .A .
1777-1784
Pergamon Press
INHIBITION OF PHFNYi .erexINE HYDROXYLASE ACTIVITY BY a(-METHYL TYROSINE, A POTENT INHIBITOR OF TYROSINE HYDRORYLASE Leela I. Murthy* Children's Hospital Research Foundation and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
45229
(Received in final form November 3, 1975) SUMMARY Inhibitors of phenylalanine hydrorylase and tyrosine hydrorylase were used in the assay of phenylalanine hydrorylase in liver and kidney of rate and mice . Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyro sine methyl ester showed 5-15X inhibition while d~methyl tyrosine seemed to inhibit phenylalanine hydrorylase to the eatent of 95-98X at concentrations of 5 x 10 -5M - 1 x 10 -4M . After a phenylketonuric diet (0 .12X PCPA + 3X excess phenylalanine), the liver ahówed -60X phénylalanine hyd=orylase activüy and 7cidney 82X that present in pair-fed normals . Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63X of normal activity . The PCPAfed animals showed 34X in liver and 38X in kidney as compared to normals ; in both cases normal activity was noticed after refeeding . The phenylalanine-fed animals showed activity similar to that seen in plteaylketonuric animals. The temporary inducement of phenylketonuric in these animals may be due to a slight change in conformation of the phenylalanine hydrorylase molecule ; once the normal diet is resumed, the enzyme reverts back to its active form . This paper also suggests thataC~methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydrorylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonnrics .
Supported by grant HD06018, from the National Institutes of Health, U. S . Public Health Service. *Present Address ; Criteria Development Branch, National Institute for Occupational Safety and Health, Post-0ffice Building, Cincinnati, Ohio 45202. Abbreviations: PKU ~ phenylketonuria or phenylketonuric ; PHE ~ phenylalanine ; TYR ~ tyrosine ; PAH = phenylalanine hydrorylase ; TYH a tyrosine hydrorylase ; PCPA = p-chlorophenylalanine ; AMT ~ OC--methyl tyrosine ; NE ~ norepinephrine ; CA ~ catecholamire(~ ;DOPA - díhydroxyphenylalanine .
1777
1778
Phenylalanine Hydroxylase Inhibition
Vol . 17, No . 12
Phenylketonuria (PKU~ which is associated with mental retardation, is an excellent study of the relation between genetics, biochemistry and behavior . Initial observations in rats (1,2) were made so as to induce conditions similar to that seen in phenylketonuric children . This led to various biochemical (3-8) and behavioral (9,10) studies . The levels of phenylalanine (PHE) and tyrosine (TYR) were studied in control rata and those fed with PHE alone or along with p-chlorophenylalanine (PCPA) . Koe and Weissman (11) observed that the in vivó administration of PCPA produced a marked inhibition of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TYH) . Lipton et al (8) demonstrated that PCPA could be used to induce the biochemical symptoms of PK[J in rats including the high serum PHE/TYR ratios which is characteristic of the human condition . Boggs and Waisman (6) made growing rata phenylketonuric by feeding excess phenylalanine . In our laboratory, Butcher et al (9)succeeded in inducing the biochemical manifestations of PKU (elevated serum PHE, elevated PHE/TYR ra~ios,,excretion of phenylpyruvic acid) when they fed rate the PKU diet consisting of 0.12X PCPA and 3X excess PHE; in addition, a learning impairment was produced in the offspring . Later, they observed hypoactivity in open field performance of rats fed the PKU diet (10) . Recently, Valdivieso et al (12) have reported the successful inhibition of PAH by PHE, PCPA and Esculin (6,7-dihydroxycoumarin 6glucoside) resulting in an enhancement of the levels of liver PHE and its metabolites, which reach values similar to those found in tissues of PRU patients while TYR concentrations remain near the control values . Tyrosine hydroxylase (TYH) appears to be the rate-limiting step in norNagatsu et al (14) observed that d-methyl epinephrine (NE) biosynthesis (13) . tyrosine (AMT) was a competitive inhibitor to TYH with a low Rm and a very low Vmax . CC -methyl tyrosine and its methyl ester have been the inhibitors widely used to demonstrate the effects of exercise, stress and various drugs on the turnover of catecholamines and also to lower NE formation in patients with pheochromocytoma and malignant hypertension (15) . Ayling and Helfand (16) observed that the reduction in inhibition of PAH was due to the synthetic cofactor used in the assay . When the natural cofactor, ~trahydrobiopterin, was used, they observed complete inhibition of PAH. Since the plasma levels of TYR were found to be higher than normal in PKU-fed rats, the present work was undertaken to see if an inhibitor of TYH would be effective in inhibiting PAH . If such an inhibitor is found, it could be fed along with the usual PKU diet and both biochemical and behavioral studies undertaken . The present work describes the in vivo and in vitro studies of inhibitors of both PAH and TYH on the activity of PAH in liver and kidney in rat and mouse. Materials and Methods Sprague-Dawley rats were obtained from Laboratory Supplies, Indianapolis, Indiana, and Ajax mice from Jackson Laboratory, Bar Harbor, Maine. L-phenylalanine and dithiothreitol were purchased from Nutritional Biochemicals, nico tine adenine dinucleotide and nicotinamide from Sigma Chemical Co ., 2-amino-4hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterine hydrochloride from Calbiochem, and L-(3- 14C) phenylalanine, ap . act . lOmCi/mmol e from Amersham Searle . Omnifluor was obtained from New England Nuclear . Inhibitors DL-p-chlorophenylalanine (PCPA) was purchased from Sigma Chemical Co ., DL~methyltyrosine, DL-oC-methyltyroaine methyl eater hydrochloride and DLa-3-dimethyl tyrosine methyl ester hydrochloride were obtained from Aldrich Chemical Co .
Vol. 17, No . 12
Phenylalanine Hydroaylaea Inhibition
1779
Tissues were removed quickly, frozen on dry ice until completion of each operation, weighed and homogenized using glass homogenizera in 3 and 6 volumes of potassium chloride (0 .15M) for kidney and liver respectively . Details of the procedure and assay system used are the same as described earlier (17,18) . In Vivo Inhibition Experiments Sprague-Dawley male rats were assigned at 21 days of age (weaning) to each of the following diet groups for 30 days : 1) PRU group - Purina Rat Chow supplemented with 3X excess PHE and 0.12X PCPA ad lib ; 2) PHE group - Purina Rat Chow supplemented with 3X excess PHE in the amount consumed each day by a matched subject in diet group (1) ; 3) PGPIL group - Purina Rat Chow supplemented with 0.12X PCPA in the amount consumed each day by a matched subject in diet group (1) ; 4) PFN group - Unaupplemented Purina Rat Chow in the amount consumed each day by a matched subject in group (1) . All animals were housed individually in quarters with alternating 12 hour periods of light and dark ; temperature was maintained at 22°f 2~, After 30 days of special diet feeding, the animals in each group were further divided into 3 sub-groups, (a) through (c) . Subgroup (a) was used for in vitro assay experiment without any further changes; subgroup (b) was fed ad lib with unaupplemented Purina Rat Chow for a period of 4 days, then sacrificed for in vitro assay, and (c) was fed ad lib for 8 days before being sacrificed for in vitro assay . Three animals were used in each phase of the experiment, In Vitro Inhibition Experiments Stock solutions of 103M and 104!i concentrations of the inhibitors were first made ; 20 and 40~u1s were used in the inhibition eaperimente o yield the following final concentrations : 1 x 10-4M, 5 a 10-5M, and 5 x 10-~. The stand ard assay was run using 20 and 40 pl of the inhibitor solution with an equivalent decrease in phosphate buffer . 6 animals were used in each experiment . Results In our assay for determining PAH activity using the radioactivity method (17,19), DOPA formation upto 1 .25X in mouse liver and up to 1.58X in mouse kidney was noticed ; however, the amount of DOPA formed in rat liver and kidney tissues was much lower (0 .5 and O.SX respectively) . The inhibitors used in this aeries were of two kinds ; a) p-chlorophenylalanine, and b) a few inhibitors of tyrosine hydroxylase (¢methyl tyroeine,oc~ethyl tyrosine methyl ester hydrochloride and d-dimethyl tyrosine methyl ester hydrochloride), Ikeda et al (20) have shown that tyrosine hydroxylase cataly9es the aromatic hydroxylation of both L-phenylalanine and L-tyrosine to DOPA . They have also shown that rat liver preparations do not contain tyrosine hydroaylase . Small amounts of DOPA have been noticed in our assays in both liver and kidney tissues . It may be that the non-enzymatic hydroxylation of phenylalaaine to tyrosine by the synthetic cofactor contributes to further hydroxylation ; in other words, tyrosine also undergoes non-enzymatic hydroxylation to DOPA which may account for the small amount of DOPA formed in our assays . Nagateu et al (14) also observed non-enzymatic hydroxylation of tgroeine to DOPA . In Vi tro Experimenta Our in vitro inhibition experiments were performed using mouse tissues . Since we do not know whether mouse liver preparations contain tyrosine hydroxylase activity, we decided to use inhibitiore of tyrosine hydroxylase (methyl
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Phenylalanine Hydroxylase Inhibition
Vol . 17, No . 12
tyrosine and its methyl esters) in order to decrease the amount of DOPA formed . Figure 1 shows the effect of various concentrations of the inhibitors used in the assay of phenylalanine hydroxylase . While the inhibition 3y PCPA, methyl tyrosine methyl ester and dimethyl tyrosine methyl ester remains between 5-15X for the concentrations 5 x 10-6M - 1 x 10 -~+M,oC-methyl tyrosine, known to be a competitive inhibitor of tyrosine hydroxylase, seems to inhibit phenylalanine hydroxylase to the extent of 98X at the concentrations 5 x 10 -5li - 1 10 -4M, and 26X inhibition at 5 x 1Q"~M concentration . In Vivo Experiments In vivo inhibition eaperimenta were performed with adult male rata (SpragueDawley) . The animals were grouped in threes, fed special diets for 30 days, then refed regular ~üet (ad libifum) for 4 and.8 days . In_. .vitro assays were d run on the liver and kidney tissues using the synthetic cofactor . ncubation at 25 for 20 minutes and'at 37 ° for 10 minutes yielded comparable phenylalanine hydroxylase activities, but when calculated per minute, the activity at 37 ° was twice that seen at 25°. Table 1 shows phenylalanine hydroxylase activity at both 25° and 37 ° . Assays for the refed animals 4 days after the diet at 37° were not performed . Looking at the activity of phenylalanine hydroxylase at 25°, in the PRU group, after the 30 day diet, the liver shows 60X activity and the kidney 82X activity that present in pair-fed normals. After 4 days of normal diet, the level of activity in both liver and kidney tissues continued to fall as However, after 8 days compared to the normal to 42X and 61X respectively . normal diet, the liver showed normal activity whereas the kidney was still 63X of normal . In the PCPA-fed animals, after the 30-day diet, the activity ín liver was 34X that present in pair-fed animals, and in the kidney 38X . In both cases the activity was normal after 8 days normal diet . The 3X phenylalanine-fed animals showed 59X activity in liver and 85X in kidney as compared to pair-fed normals . Discussion Parachlorophenylalanine, a potent in vivo inhibitor of phenylalanine hydroxylase has been used in conjunction with phenylalanine to simulate phenylketonuria in rate (6,8,9,10,11) for the in vitro studies using both natural and synthetic cofactor . The use of PCPA together with PHE in vivo produces marked elevation of plasma phenylalanine as in seen in the human disease phenylketonuria . However, the experimental models fai .1 to mimic It the human defect which shows normal levels of tyrosine in the plasma . may be that, experimentally, phenylalanine as well as some of the appropriate aromatic urinary metabolites are elevated as seen in hyperphenylalaninemia, with the exception of the enzyme not being completely inhibited . This results in partial conversion of phenylalanine to tyrosine thereby yielding elevated levels of TYR in addition to that of PHE and its alternate metabolites. Thus, in addition to PCPA and 3X PHE in the diet, another inhibitor that would inhibit PAH completely may be needed . Ia the present work, both in vitro and in vivo experiments were performed using different inhibitors . In the in vitro experiments, inhihiro±'n of ~+oth phenylalanine hydroxylase and tyrosine hydroxylase were etucjied . In the liver, PCPA showed only 5-15X inhibition in concentrations 5 x 10 1 x 10 -4M. This is in agreement with the findings of Ayling and Helfaad (16) who observed that the inhibition of phenylalanine hydroxylase by PCPA was low with synthetic cofactor as compared to that with the natural cofactor . In addition, our studies reveal that a-methyl tyrosine, a competitive inhibitor of tyrosine hydroxy-
Phenylalanine Hydroxylase Inhibition
Vol . 15, No . 12
1781
100
80
60
40
20
oirrrE 5xI0~M
5x10' °M
I x 10' 4 M
INHIBITOR CONCENTRATION FIG . 1 Inhibition of phenylalanine hydroxylase activity by p-chlorophenylalanine (PCPA),oCrmethyl tyrosine (AMT), a-methyl tyrosine methyl ester hydrochloride (MTME) andec-dimethyl tyrosine methyl eater hydrochloride (DMTME) . The assay system contained 0 .08 y~ole NAD, 1 .0 ymole nicotinamide, 0 .27 pmole 2-amino-4hydroxy-ó,7-dimethyl-5,6,7,8-tetrahydropteridine in 0.1 M dithiothreitol (2 .0 Nmoles), O .1,uCi L-(14C) phenylalanine, 1 .O,umole L-phenylalanine for assay of kidney supernatant, and 2 .0 pmoles for liver supernatant, 0.1 ml supernatant containing the enzyme and 0.2 M phosphate, pN 7 .0, to give a final volume of 0.4 ml . Incubation at 25°C was for 20 min . Six animals were used in each experiment .
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Phenylalanine Hydrozvylase Inhibition
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17, No . 12
TABLE 1 PHENYLALANINE HYDROXYLASE ACTIVITY IN LIVER & KIDNEY OF RATS FED SPECIAL DIETS LIVER
KIDNEY
~unoles Tyrosine /min/g tissue PFN
PKU
PCPA
PHE
.317
.191
.107
.188
.284
.120
-
.268
.315
.27 1
30 day diet
.624
.380
.159
8 days ad lib after 30 d .diet
.640
.677
.62 1
_25 0 30 day diet 4 days ad lib after 30 d .diet 8 days ad lib after 30 d .diet
ymoles Tyrosine /min/g tissue PFN
PKU
PCPA
PHE
.103
.084
.039
.088
-
.112
.068
-
-
.096
.061
.104
.197
.163
.069
.173
.200
.128
.184
-
370 .429 -
- not recorded Diets : PFN = pair-fed normal ; PKU = 0 .12% p-chloro-phenylalanine + excess phenylalanine ; PCPA = 0 .12% p-chloro-phenylalanine ; PHE = 3% excess phenylalanine Three animals were used in each phase of experiment . The assay system used is the same as described in Fig . 1 .
3%
lass, is also an inhibitor of hep tic phenyls same hydroxylase in the cor_centration ra_ige studied (5 a 10 ~ - 1 g 10 ) . In the few experiments on kidney tissue, the inhibitio of PAH activity by both PCPA and AMT was 15-27% at concentrations of 5 a 10 -~ - 1 a 10 -~! . It may be of interest to see if eC-methyl tyrosine in conjunction with PCPA and PAE would resemble human phenylketonuric conditions with elevated levels of phenylalanine, but normal tyrosine levels . In our in vivo eaperimente, it has been noticed that the activity of phenylalanine hydroxylase ie lessened after a 30-day PKU diet and remains low even after 4 d$ys of normal diet . However, continuation of refeeding with normal diet for 8 days brings the level of activity of phenylalanine hydroxylase back to normal . In other words, the inducement of phenylketonuria in these experimental animals by PCPA and PHE does not completely inhibit phenylalanine hydroxylase, but may be changing the conformation of the hydroxylase enzyme to a certain extent, thus affecting the active site . Once the extraneous addition of PCPA and PHE is withdrawn and normal diet is resumed the phenylalanine hydroxylase molecule reverts back to its active form~yi.elding normal amounts of phenyl alanine hydroxylase activity . We may ales suggest that addition of oc-methyl tyrosine to the diet containing PCPA and PHE may result in complete inhibition of the phenylalanine hydroxylase enzyme resulting in elevated levels of phenylalanine, but normal levels of tyrosine . It may also be worthwhile to determine if the levels of serotonin are decreased as is seen in pheaylketonuric children and in cases where tyrosine hydroxylase is inhibited . Our acknowlédgmenta to Dr . R . Butcher and his assistants for the assignment and feeding of rats with the special diets.
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Phenylalanine Hydroxylase Inhibition
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References 1.
V. H . Auerbach, H. A. Waisman and L. B . Wyckoff Jr ., Nature 182 : 871 -872 (1958) .
2.
H. L . Wang and H. A. Waieman, Proc . Soc. Exptl . Biol . Med. _108 : 332-335 (1961) .
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A. Yuwiller and R. T . Louttit, Science 134 :831-832 (1961) .
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C . M. McKean, S. M. Schanberg, and N. J. 604-605 (1962) .
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I . Huang and D . Y . Y . Heia, Proc . Soc. Exptl . Biol . Med . (1963) .
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D . E. Boggs and H . A. Waieman, Arch . Biochem. Biophys, (1964) .
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D . E. Boggs and H . A. Waisman, Proc . Soc. Exptl. Biol . Med . 407-410 (1964) .
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M . A. Lipton, R . Gordon, G . Guroff and S . Udenfriend, Science _156 : 248-250 (1967) .
9.
R . Butcher, C . Vorhees and H. R . Berry, Life Sci. 9 :1261-1268 (1970) .
Giarman, Science _i37 : _112 :81-84
106 : 307-311 _i15 :
10 .
C . V. Vorheea, R. E, 5 :175-179 (1972) .
Butcher and H. R. Berry, Develop . Psychobiol .
11 .
B . K. Roe and A . Weismann, J. Pharmacol. Exp . Ther . 154 :499-516 (1966) .
12 .
F . Valdivieao, Biochem. Med . 12 :72-78 (1975) .
13 .
M . Levitt, S . Spector, A. Sjoerdsma and S . Udenfriend, J . Pharmacol.
14 .
T. Nagateu, M . Levitt and S . Udenfriend, J . Biol . Chem ., (1964) .
15 .
J . R. Cooper, F . E. Bloom, R. H . Roth, Biochemical Basica of Neuropharmacology . p . 112, Oxford University Prese, London (1974) .
16 .
J . E. Ayling and G. D . Helfand 366 (1974) .
17 .
L . I. Murthy and H . K. Berry, Arch . Biochem . Biophys., 163 :225-230 (1974)
18 .
H. K. Berry, R. Crippa, R. Nicholls,D . McCandless and C . Harper ., Biochem . Biophys, Acta, 261 :315-320 (1972)
19 .
L . I . Murthy and H. K. Berry, Biochem . Med., 12 :392-397
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M . Ikeda, M. Levitt and S . Udenfriend, Biochem, Biophys . Rèa . Comm ., 18 :482-488 (1965) .
239 :2910-2917
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(1975) .
1777-1784
Pergamon Press
INHIBITION OF PHFNYi .erexINE HYDROXYLASE ACTIVITY BY a(-METHYL TYROSINE, A POTENT INHIBITOR OF TYROSINE HYDRORYLASE Leela I. Murthy* Children's Hospital Research Foundation and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
45229
(Received in final form November 3, 1975) SUMMARY Inhibitors of phenylalanine hydrorylase and tyrosine hydrorylase were used in the assay of phenylalanine hydrorylase in liver and kidney of rate and mice . Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyro sine methyl ester showed 5-15X inhibition while d~methyl tyrosine seemed to inhibit phenylalanine hydrorylase to the eatent of 95-98X at concentrations of 5 x 10 -5M - 1 x 10 -4M . After a phenylketonuric diet (0 .12X PCPA + 3X excess phenylalanine), the liver ahówed -60X phénylalanine hyd=orylase activüy and 7cidney 82X that present in pair-fed normals . Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63X of normal activity . The PCPAfed animals showed 34X in liver and 38X in kidney as compared to normals ; in both cases normal activity was noticed after refeeding . The phenylalanine-fed animals showed activity similar to that seen in plteaylketonuric animals. The temporary inducement of phenylketonuric in these animals may be due to a slight change in conformation of the phenylalanine hydrorylase molecule ; once the normal diet is resumed, the enzyme reverts back to its active form . This paper also suggests thataC~methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydrorylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonnrics .
Supported by grant HD06018, from the National Institutes of Health, U. S . Public Health Service. *Present Address ; Criteria Development Branch, National Institute for Occupational Safety and Health, Post-0ffice Building, Cincinnati, Ohio 45202. Abbreviations: PKU ~ phenylketonuria or phenylketonuric ; PHE ~ phenylalanine ; TYR ~ tyrosine ; PAH = phenylalanine hydrorylase ; TYH a tyrosine hydrorylase ; PCPA = p-chlorophenylalanine ; AMT ~ OC--methyl tyrosine ; NE ~ norepinephrine ; CA ~ catecholamire(~ ;DOPA - díhydroxyphenylalanine .
1777
1778
Phenylalanine Hydroxylase Inhibition
Vol . 17, No . 12
Phenylketonuria (PKU~ which is associated with mental retardation, is an excellent study of the relation between genetics, biochemistry and behavior . Initial observations in rats (1,2) were made so as to induce conditions similar to that seen in phenylketonuric children . This led to various biochemical (3-8) and behavioral (9,10) studies . The levels of phenylalanine (PHE) and tyrosine (TYR) were studied in control rata and those fed with PHE alone or along with p-chlorophenylalanine (PCPA) . Koe and Weissman (11) observed that the in vivó administration of PCPA produced a marked inhibition of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TYH) . Lipton et al (8) demonstrated that PCPA could be used to induce the biochemical symptoms of PK[J in rats including the high serum PHE/TYR ratios which is characteristic of the human condition . Boggs and Waisman (6) made growing rata phenylketonuric by feeding excess phenylalanine . In our laboratory, Butcher et al (9)succeeded in inducing the biochemical manifestations of PKU (elevated serum PHE, elevated PHE/TYR ra~ios,,excretion of phenylpyruvic acid) when they fed rate the PKU diet consisting of 0.12X PCPA and 3X excess PHE; in addition, a learning impairment was produced in the offspring . Later, they observed hypoactivity in open field performance of rats fed the PKU diet (10) . Recently, Valdivieso et al (12) have reported the successful inhibition of PAH by PHE, PCPA and Esculin (6,7-dihydroxycoumarin 6glucoside) resulting in an enhancement of the levels of liver PHE and its metabolites, which reach values similar to those found in tissues of PRU patients while TYR concentrations remain near the control values . Tyrosine hydroxylase (TYH) appears to be the rate-limiting step in norNagatsu et al (14) observed that d-methyl epinephrine (NE) biosynthesis (13) . tyrosine (AMT) was a competitive inhibitor to TYH with a low Rm and a very low Vmax . CC -methyl tyrosine and its methyl ester have been the inhibitors widely used to demonstrate the effects of exercise, stress and various drugs on the turnover of catecholamines and also to lower NE formation in patients with pheochromocytoma and malignant hypertension (15) . Ayling and Helfand (16) observed that the reduction in inhibition of PAH was due to the synthetic cofactor used in the assay . When the natural cofactor, ~trahydrobiopterin, was used, they observed complete inhibition of PAH. Since the plasma levels of TYR were found to be higher than normal in PKU-fed rats, the present work was undertaken to see if an inhibitor of TYH would be effective in inhibiting PAH . If such an inhibitor is found, it could be fed along with the usual PKU diet and both biochemical and behavioral studies undertaken . The present work describes the in vivo and in vitro studies of inhibitors of both PAH and TYH on the activity of PAH in liver and kidney in rat and mouse. Materials and Methods Sprague-Dawley rats were obtained from Laboratory Supplies, Indianapolis, Indiana, and Ajax mice from Jackson Laboratory, Bar Harbor, Maine. L-phenylalanine and dithiothreitol were purchased from Nutritional Biochemicals, nico tine adenine dinucleotide and nicotinamide from Sigma Chemical Co ., 2-amino-4hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterine hydrochloride from Calbiochem, and L-(3- 14C) phenylalanine, ap . act . lOmCi/mmol e from Amersham Searle . Omnifluor was obtained from New England Nuclear . Inhibitors DL-p-chlorophenylalanine (PCPA) was purchased from Sigma Chemical Co ., DL~methyltyrosine, DL-oC-methyltyroaine methyl eater hydrochloride and DLa-3-dimethyl tyrosine methyl ester hydrochloride were obtained from Aldrich Chemical Co .
Vol. 17, No . 12
Phenylalanine Hydroaylaea Inhibition
1779
Tissues were removed quickly, frozen on dry ice until completion of each operation, weighed and homogenized using glass homogenizera in 3 and 6 volumes of potassium chloride (0 .15M) for kidney and liver respectively . Details of the procedure and assay system used are the same as described earlier (17,18) . In Vivo Inhibition Experiments Sprague-Dawley male rats were assigned at 21 days of age (weaning) to each of the following diet groups for 30 days : 1) PRU group - Purina Rat Chow supplemented with 3X excess PHE and 0.12X PCPA ad lib ; 2) PHE group - Purina Rat Chow supplemented with 3X excess PHE in the amount consumed each day by a matched subject in diet group (1) ; 3) PGPIL group - Purina Rat Chow supplemented with 0.12X PCPA in the amount consumed each day by a matched subject in diet group (1) ; 4) PFN group - Unaupplemented Purina Rat Chow in the amount consumed each day by a matched subject in group (1) . All animals were housed individually in quarters with alternating 12 hour periods of light and dark ; temperature was maintained at 22°f 2~, After 30 days of special diet feeding, the animals in each group were further divided into 3 sub-groups, (a) through (c) . Subgroup (a) was used for in vitro assay experiment without any further changes; subgroup (b) was fed ad lib with unaupplemented Purina Rat Chow for a period of 4 days, then sacrificed for in vitro assay, and (c) was fed ad lib for 8 days before being sacrificed for in vitro assay . Three animals were used in each phase of the experiment, In Vitro Inhibition Experiments Stock solutions of 103M and 104!i concentrations of the inhibitors were first made ; 20 and 40~u1s were used in the inhibition eaperimente o yield the following final concentrations : 1 x 10-4M, 5 a 10-5M, and 5 x 10-~. The stand ard assay was run using 20 and 40 pl of the inhibitor solution with an equivalent decrease in phosphate buffer . 6 animals were used in each experiment . Results In our assay for determining PAH activity using the radioactivity method (17,19), DOPA formation upto 1 .25X in mouse liver and up to 1.58X in mouse kidney was noticed ; however, the amount of DOPA formed in rat liver and kidney tissues was much lower (0 .5 and O.SX respectively) . The inhibitors used in this aeries were of two kinds ; a) p-chlorophenylalanine, and b) a few inhibitors of tyrosine hydroxylase (¢methyl tyroeine,oc~ethyl tyrosine methyl ester hydrochloride and d-dimethyl tyrosine methyl ester hydrochloride), Ikeda et al (20) have shown that tyrosine hydroxylase cataly9es the aromatic hydroxylation of both L-phenylalanine and L-tyrosine to DOPA . They have also shown that rat liver preparations do not contain tyrosine hydroaylase . Small amounts of DOPA have been noticed in our assays in both liver and kidney tissues . It may be that the non-enzymatic hydroxylation of phenylalaaine to tyrosine by the synthetic cofactor contributes to further hydroxylation ; in other words, tyrosine also undergoes non-enzymatic hydroxylation to DOPA which may account for the small amount of DOPA formed in our assays . Nagateu et al (14) also observed non-enzymatic hydroxylation of tgroeine to DOPA . In Vi tro Experimenta Our in vitro inhibition experiments were performed using mouse tissues . Since we do not know whether mouse liver preparations contain tyrosine hydroxylase activity, we decided to use inhibitiore of tyrosine hydroxylase (methyl
1780
Phenylalanine Hydroxylase Inhibition
Vol . 17, No . 12
tyrosine and its methyl esters) in order to decrease the amount of DOPA formed . Figure 1 shows the effect of various concentrations of the inhibitors used in the assay of phenylalanine hydroxylase . While the inhibition 3y PCPA, methyl tyrosine methyl ester and dimethyl tyrosine methyl ester remains between 5-15X for the concentrations 5 x 10-6M - 1 x 10 -~+M,oC-methyl tyrosine, known to be a competitive inhibitor of tyrosine hydroxylase, seems to inhibit phenylalanine hydroxylase to the extent of 98X at the concentrations 5 x 10 -5li - 1 10 -4M, and 26X inhibition at 5 x 1Q"~M concentration . In Vivo Experiments In vivo inhibition eaperimenta were performed with adult male rata (SpragueDawley) . The animals were grouped in threes, fed special diets for 30 days, then refed regular ~üet (ad libifum) for 4 and.8 days . In_. .vitro assays were d run on the liver and kidney tissues using the synthetic cofactor . ncubation at 25 for 20 minutes and'at 37 ° for 10 minutes yielded comparable phenylalanine hydroxylase activities, but when calculated per minute, the activity at 37 ° was twice that seen at 25°. Table 1 shows phenylalanine hydroxylase activity at both 25° and 37 ° . Assays for the refed animals 4 days after the diet at 37° were not performed . Looking at the activity of phenylalanine hydroxylase at 25°, in the PRU group, after the 30 day diet, the liver shows 60X activity and the kidney 82X activity that present in pair-fed normals. After 4 days of normal diet, the level of activity in both liver and kidney tissues continued to fall as However, after 8 days compared to the normal to 42X and 61X respectively . normal diet, the liver showed normal activity whereas the kidney was still 63X of normal . In the PCPA-fed animals, after the 30-day diet, the activity ín liver was 34X that present in pair-fed animals, and in the kidney 38X . In both cases the activity was normal after 8 days normal diet . The 3X phenylalanine-fed animals showed 59X activity in liver and 85X in kidney as compared to pair-fed normals . Discussion Parachlorophenylalanine, a potent in vivo inhibitor of phenylalanine hydroxylase has been used in conjunction with phenylalanine to simulate phenylketonuria in rate (6,8,9,10,11) for the in vitro studies using both natural and synthetic cofactor . The use of PCPA together with PHE in vivo produces marked elevation of plasma phenylalanine as in seen in the human disease phenylketonuria . However, the experimental models fai .1 to mimic It the human defect which shows normal levels of tyrosine in the plasma . may be that, experimentally, phenylalanine as well as some of the appropriate aromatic urinary metabolites are elevated as seen in hyperphenylalaninemia, with the exception of the enzyme not being completely inhibited . This results in partial conversion of phenylalanine to tyrosine thereby yielding elevated levels of TYR in addition to that of PHE and its alternate metabolites. Thus, in addition to PCPA and 3X PHE in the diet, another inhibitor that would inhibit PAH completely may be needed . Ia the present work, both in vitro and in vivo experiments were performed using different inhibitors . In the in vitro experiments, inhihiro±'n of ~+oth phenylalanine hydroxylase and tyrosine hydroxylase were etucjied . In the liver, PCPA showed only 5-15X inhibition in concentrations 5 x 10 1 x 10 -4M. This is in agreement with the findings of Ayling and Helfaad (16) who observed that the inhibition of phenylalanine hydroxylase by PCPA was low with synthetic cofactor as compared to that with the natural cofactor . In addition, our studies reveal that a-methyl tyrosine, a competitive inhibitor of tyrosine hydroxy-
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100
80
60
40
20
oirrrE 5xI0~M
5x10' °M
I x 10' 4 M
INHIBITOR CONCENTRATION FIG . 1 Inhibition of phenylalanine hydroxylase activity by p-chlorophenylalanine (PCPA),oCrmethyl tyrosine (AMT), a-methyl tyrosine methyl ester hydrochloride (MTME) andec-dimethyl tyrosine methyl eater hydrochloride (DMTME) . The assay system contained 0 .08 y~ole NAD, 1 .0 ymole nicotinamide, 0 .27 pmole 2-amino-4hydroxy-ó,7-dimethyl-5,6,7,8-tetrahydropteridine in 0.1 M dithiothreitol (2 .0 Nmoles), O .1,uCi L-(14C) phenylalanine, 1 .O,umole L-phenylalanine for assay of kidney supernatant, and 2 .0 pmoles for liver supernatant, 0.1 ml supernatant containing the enzyme and 0.2 M phosphate, pN 7 .0, to give a final volume of 0.4 ml . Incubation at 25°C was for 20 min . Six animals were used in each experiment .
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TABLE 1 PHENYLALANINE HYDROXYLASE ACTIVITY IN LIVER & KIDNEY OF RATS FED SPECIAL DIETS LIVER
KIDNEY
~unoles Tyrosine /min/g tissue PFN
PKU
PCPA
PHE
.317
.191
.107
.188
.284
.120
-
.268
.315
.27 1
30 day diet
.624
.380
.159
8 days ad lib after 30 d .diet
.640
.677
.62 1
_25 0 30 day diet 4 days ad lib after 30 d .diet 8 days ad lib after 30 d .diet
ymoles Tyrosine /min/g tissue PFN
PKU
PCPA
PHE
.103
.084
.039
.088
-
.112
.068
-
-
.096
.061
.104
.197
.163
.069
.173
.200
.128
.184
-
370 .429 -
- not recorded Diets : PFN = pair-fed normal ; PKU = 0 .12% p-chloro-phenylalanine + excess phenylalanine ; PCPA = 0 .12% p-chloro-phenylalanine ; PHE = 3% excess phenylalanine Three animals were used in each phase of experiment . The assay system used is the same as described in Fig . 1 .
3%
lass, is also an inhibitor of hep tic phenyls same hydroxylase in the cor_centration ra_ige studied (5 a 10 ~ - 1 g 10 ) . In the few experiments on kidney tissue, the inhibitio of PAH activity by both PCPA and AMT was 15-27% at concentrations of 5 a 10 -~ - 1 a 10 -~! . It may be of interest to see if eC-methyl tyrosine in conjunction with PCPA and PAE would resemble human phenylketonuric conditions with elevated levels of phenylalanine, but normal tyrosine levels . In our in vivo eaperimente, it has been noticed that the activity of phenylalanine hydroxylase ie lessened after a 30-day PKU diet and remains low even after 4 d$ys of normal diet . However, continuation of refeeding with normal diet for 8 days brings the level of activity of phenylalanine hydroxylase back to normal . In other words, the inducement of phenylketonuria in these experimental animals by PCPA and PHE does not completely inhibit phenylalanine hydroxylase, but may be changing the conformation of the hydroxylase enzyme to a certain extent, thus affecting the active site . Once the extraneous addition of PCPA and PHE is withdrawn and normal diet is resumed the phenylalanine hydroxylase molecule reverts back to its active form~yi.elding normal amounts of phenyl alanine hydroxylase activity . We may ales suggest that addition of oc-methyl tyrosine to the diet containing PCPA and PHE may result in complete inhibition of the phenylalanine hydroxylase enzyme resulting in elevated levels of phenylalanine, but normal levels of tyrosine . It may also be worthwhile to determine if the levels of serotonin are decreased as is seen in pheaylketonuric children and in cases where tyrosine hydroxylase is inhibited . Our acknowlédgmenta to Dr . R . Butcher and his assistants for the assignment and feeding of rats with the special diets.
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