- Email: [email protected]
A study of the development of phenylalanine hydroxylase in fetuses of several mammalian species
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
146, 321-326 (1971)
A Study of the Development of Phenylalanine Hydroxylase in Fetuses of Several Mammalian Species P A U L A. F R I E D M A N AND S E Y M O U R K A U F M A N Laboratory of Neurochemistry, National Inslituge of Mental Heallh, Beghesda, Maryland 2001~ Received May 17, 1971; accepted June 11, 1971 The developmental pattern of hepatic phenylManine hydroxylase has been studied in several different mammals. Two strikingly different patterns have been observed. In the rat, no activity can be detected until the day before birth. The hydroxylase activity of newborn rats is equal to or slightly higher than that of adults. In the guinea pig and the human, in contrast, hydroxylase activity is detectable in the liver during the second third of the gestation period. I t is now well established that, the enzymatic conversion of phenylalanine to tyrosine in m a m m a l i a n liver is catalyzed b y a complex system consisting of several enzymes and eoenzymes. The minimum requirements are for phenylalanine hydroxylase and a tetrahydropterin eofaetor (1). The eofaetor requirement can be filled by either the naturally occurring compound, tetrahydrobiopterin (2), or b y one of several synthetic analogs, such as 6,7-dimethyltetrahydropterin (3): In the presence of catalytic concentrations of the pterin cofactor, the hydroxylation reaction is also dependent on a tetrahydropterin-regenerating system, such as dihydropteridine reductase and T P N H (4). I n 1958 (5, 6), it, was reported t h a t phenylalanine hydroxylase was a m e m b e r of a group of m a m m a l i a n liver enzymes t h a t are essentially inactive at birth (7-11). The activities of these enzymes increase rapidly after birth, usually reaching the adult levels within 1 or 2 days (7-11). I n 1965, we showed t h a t phenylalanine hydroxylase did not belong to this group of enzymes t h a t are inactive at birth. I t was found t h a t when properly supplemented with dihydropteridine reductase and pterin cofaetor, the hydroxylase activity in livers from newborn rats is equal to, or higher than, t h a t of adults (12). The levels of the pterin
cofactor and of dihydropteridine reductase in the newborn livers were relatively deficient (12). A study of the development of phenylalanine hydroxylase activity in fetal chicken liver showed t h a t the activity appeared during the second half of the incubation period and approached adult levels at t e r m (13). I n the present paper, we report the results of studies on the deve]opmentM pattern of phenylalanine hydroxylase in the fetal livers of several m a m m a l i a n species. Two strikingly different patterns have been observed. I n the guinea pig and the human, hydroxylase activity is detectable in fetal livers during the second third of the gestation period. B y contrast, in the rat, no activity is detected until the day before birth. MATERIALS AND METHODS
Sprague-Dawley rats and Hartley guinea pigs were obtained from the National Institutes of Health animal supply section. Liver tissue from a 23-week-old human fetus was kindly supplied by Dr. Neil Holtzman of Johns Hopkins Hospital, Baltimore, Md. The liver was removed from the fetus within 1 hr of a therapeutic abortion and was quick frozen and stored at -80 ~. The activity was measured after 4 weeks of frozen storage. Tyrosine and phenylalanine were obtained from NutritionM Biochemicals. TPN + was purchased from Sigma Chemical Co., and glucose-6321
322
FRIEDMAN AND KAUFMAN
phosphate from Calbioehem. Catalase, glucose-6phosphate-dehydrogenase, and TPNH were obtained from Boehringer Mannheim. 2-Amino-4hydroxy - 6,7 - dimethyl - 5,6,7,8 - tetrahydropteridine was purchased from Aldrich Chemical Co. Glucose dehydrogenase was prepared by a modification of the method by Strecker and Korkes (14); the fractions used supply adequate amounts of catalase, an enzyme that is required to protect the hydroxylating system against tI20~-mediated inactivation (15). Preparation of tissues. Fetal animMs were delivered by Cesarean section immediately after the mothers had been killed by cervical fracture. Guinea pig fetal age was approximated from fetal size and estimated date of conception. Rat fetal age was estimated from the degree of skeletal development with the use of a modification of the clearing and staining procedures of Walker and Wirtschrafter (16). Fetal and newborn animals were killed by decapitation; adult animals by cervical fracture. All steps were performed at 0-4 ~ Livers obtained from fetal or newborn animals were pooled for experiments. They were cooled and washed in 0.25M sucrose, blotted, weighed, minced, and homogenized manually in an all-glass PotterElvehjem homogenizer. Extracts for hydroxylase assays were prepared by homogenization of the livers with 3 vol of 0.15 M KC1 unless otherwise stated. When human fetal liver was to be assayed, a small, still frozen portion of tissue was removed from the bulk of the liver with a razor blade in a cold room at - 2 0 ~ Prior to homogenization, the required volume of XC1 solution was added to the frozen tissue which was then allowed to thaw at 4 ~ The homogenates were centrifuged at 16,000g for 15 min, and the supernatant fluids were immediately used to assay for phenylalanine hydroxylase or dihydropteridine reductase activities. For cofactor assays, the liver extracts were prepared as described above except that 2 vol of glass-distilled water was used for homogenization. The supernatant fluids were boiled for 2 min with stirring. They were rapidly cooled and centrifuged for 15 rain at 16,00~g. The clear yellow supernatant fluid was assayed for phenylManine hydroxylation cofactor activity (17). Enzyme assays. Phenylalanine hydroxylase was assayed according to published procedures (4, 18); the incubation period was either 30 or 45 rain. Dihydropteridine reductase was assayed by a modification of the standard phenylalanine hydroxylase assay system (4). Excess amounts of phenylalanine hydroxylase prepared from rat liver and purified through the Sephadex G-200
step were used (19). Synthetic dimethyltetrahydropterin (1) was used as the cofactor at a concentration of 0.3 mM. In all experiments, the concentration of the pterin was calculated from a measurement of its absorbance in 2 N perchlorie acid (extinction coefficient = 17.8 X 10s ~-i cm-1 at 265 mt~) (20). Tyrosine formation was measured either speetrophotometrically or fluorometricMly by the nitrosonaphthol method (21, 22). Protein was determined by the Lowry method with the use of bovine serum albumin as the standard (23). RESULTS T a b l e I shows a c o m p a r i s o n of tile act i v i t y of the p h e n y l a l a n i n e - h y d r o x y l a t i n g s y s t e m i n livers from fetal, n e w b o r n , a n d a d u l t male rats u n d e r v a r i o u s conditions. As f o u n d previously (12), w i t h o u t supplem e n t a t i o n , n e w b o r n livers h a v e significant a c t i v i t y , a l t h o u g h lower t h a n t h a t of a d u l t males. 1 T o d e t e r m i n e w h e t h e r the r e l a t i v e l y low a c t i v i t y of t h e n e w b o r n s was due to a deficiency of d i h y d r o p t e r i d i n e reductase, cofactor, or p h e n y l a l a n i n e hydroxylase, t h e s y s t e m was assayed after s u p p l e m e n t a t i o n with d i h y d r o p t e r i d i n e reductase a n d / o r cofactor. T h e results, s h o w n i n T a b l e I, are i n a g r e e m e n t with those previously r e p o r t e d (12). I n a s y s t e m s u p p l e m e n t e d with both d i h y d r o p t e r i d i n e r e d u c t a s e a n d cofactor the h y d r o x y l a s e a c t i v i t y i n n e w b o r n livers is at least equal to t h a t of adults. T h e greater s t i m u l a t i o n of t h e n e w b o r n liver extracts b y cofactor a n d d i h y d r o p t e r i d i n e r e d u c t a s e is i n a g r e e m e n t with previous o b s e r v a t i o n s (12) a n d supports the conclusion t h a t b o t h of these c o m p o n e n t s are relatively deficient i n the n e w b o r n livers (12). Similar studies with fetal livers ( T a b l e I) d e m o n s t r a t e t h a t i n t h e system supplem e n t e d with b o t h d i h y d r o p t e r i d i n e reductase a n d cofactor, no significant hydroxylase a c t i v i t y is detectable earlier t h a n 24 to 36 The hydroxylase activities of both the newborn and adult liver extracts are about 60% higher than those reported previously (12). The only known variables between the previous and present procedures are: (a) gentle hand homogenization of the livers was used in the present procedure instead of the previously used motor~driven homogelfizer, and (b) somewhat higher levels of the pterin cofactor were used in the present assays (0.30 m~ versus 0.22 m~).
323
P H E N Y L A L A N I N E H Y D R O X Y L A S E IN M A M M A L S TABLE I 1)ItENYLALANINE HYDROXYLASE
NEWBORN~
A C T I V I T I E S IN F E T A L ,
AND
ADULT
t~ATS a
Tyrosine formed Age of animalsb
No additions ~mole/ml extract
D a y 14-17 fetus Late d a y 18 fetus ]Day 21 fetus E a r l y d a y 22 fetus L a t e d a y 22 fetus L a t e d a y 22 featus (during active labor) 6-Hr n e w b o r n A d u l t males~
q- Dihydropteridine reductase
q- Dihydropteridine reductase and tetrahydropterin
-~ Tetrahydropterin
gmole/mg ~mole/ml /*mole/rag t~mole/ml amole/mg ~mole/ml ~ole/mg extract protein protein protein extract protein extract
0 0 0 0.018 0.093
0 0 0 0. 002 0.008
0 0 0 0. 045 0.113
0 0 0 0. 004 0.010
0 0 0 0.225 1.161
0 0 0 0.019 0.106
0 0 0 0.263 1.610 2.101
0 0 0 0.022 0.146 0.168
0.210 0.580 4-0.040
0.013 0.027 4-0.004
0.240 0.704 4-0.040
0.015 0.033 -t-0.003
1.143 1.452
0.072 0.068 4-0.003
2.613 2.145
0.157 0.099
•
•
•
T h e complete s y s t e m c o n t a i n e d t h e following components (in micromoles unless otherwise s t a t e d ) : p o t a s s i u m p h o s p h a t e buffer, p H 6.8, 50: L-phenylManine, 1.0; T P N +, 0.15; glucose, 125; glucose dehydrogenase, 75 milts; liver e x t r a c t , 0.2 ml. D i h y d r o p t e r i d i n e r e d u c t a s e (4.9 mg of p r o t e i n ) and dim e t h y l t e t r a h y d r o p t e r i n (0.15 ~mole) were added where indicated. T h e final volume was 0.5 nil, t h e i n c u b a t i o n t i m e was 45 min, and t h e t e m p e r a t u r e was 25 ~ F r o m each g r a m wet w e i g h t of liver 2.6 ml of e x t r a c t is obtaineff. b E a c h experiment w i t h fetal or n e w b o r n animals represents 6-12 pooled livers per litter. Results are an average of four experiments 4- s t a n d a r d deviation.
hr prior to parturition. There follows a dramatic rise in activity (Fig. 1) until late in l'~bor hydroxylase activity reaches adult levels. If fetal liver is supplemented with either dihydropteridine reductase or with cofactor, there is stimulation of hydroxylase activity. The stimulation with cofactor is much more striking and accounts for much of the effect seen in the system supplemented with both dihydropteridine reductase and cofactor. These data suggest that in fetal livers which show hydroxylase activity, cofactor levels are deficient when compared to livers of newborns and adults. The possibility that all three essential components of the phenylalanine-hydroxylating system develop together was examined by assaying livers of several fetuses of different ages for dihydropteridine reductase and for cofactor activity (Table II). It is apparent that in fetal livers which have no hydroxylase activity (day 18, day 20-21), both dihydropteridine reductase and cofactor are present. In the last 24-48 hr of fetal life, however, there is more than a
5.0 E --L i
2.0'
\
~o
0 ':t
~- 0
16 18 GESTATION AGE (Days)
FIG. 1. P h e n y l a l a n i n e hydroxylase activity in fetal, newborn, a n d adult r a t liver. This curve is a plot of the d a t a in T a b l e I in t h e system to which d i h y d r o p t e r i d i n e reductase and d i m e t h y l t e t r a h y d r o p t e r i n have been added. N = 6-hr newborn; A = adult.
twofold increase in both activities. Thus, although the appearance in fetal life of the three components of the phenyla]aninehydroxylating system is not simultaneous,
324
FRIEDMAN AND KAUFMAN TABLE II
DIHYDROPTERIDINE I:~EDUCTASE AND PHENYL ~ ALANINE ]-IyDROXYLATIO N COFACTOR ACTIVITIES IN FETAL AND ADULT I~ATSa
Experimental animals
Dihydropteridlne reductase activity (tyrosine formed) ttmole/ml extract
Day-18 fetus Day-20-21 fetus Late day-22 fetus
(during labor) Adult males b
0.115 0.135 0.300
#mole/rag protein
0.012 0.014 0.024
Cofactor activity (uaits/ml extract)
0.056 0.065 0.169
0.885 0.048 0. 532 4-0.042 =t=0.004 =t=0.043
The complete assay system for dihydropteridine reductase activity is described under Materials and Methods. The complete assay system for cofactor activity contained the stone components as described in Table I with the following exceptions: PhenylManine hydroxylase from rats (0.180 mg of protein), purified through the G-200 Sephadex step (19) and dihydropteridine reduetase from sheep (9.8 mg of protein), purified through the first ammonium sulfate precipitation (4), were used. Cofactor activity was determined on boiled extracts of liver (0.4 ml) prepared as described in Materials and Methods. In both assays, the volume was 1.0 ml and incubations were for 45 min at 25~ One unit of eofaetor activity represents the additional formation of 1.0 t,moles of tyrosine under the standard assay conditions (17). b Average of two experiments 4- range. there is a similar marked increase in the activities of phenylalanine hydroxylase, dihydropteridine reduetase, and eofaetor just prior to birth. The development of phenylalanine hydroxylase activity in fetal rats is quite different from that reported for fetal chickens (13). I n chickens, enzyme activity appears at about fetal day 12 of a 20-day incubation period and reaches approximately adult levels b y fetal day 17. To determine if the pattern in fetal rats is representative of the development of phenylalanine hydroxylase in mammals in general, guinea pig and human fetal liver were also studied. Table I I I shows that liver from a 40-45day fetal guinea pig (gestation period 64 days) already has one-tenth of the activity
of adult male guinea pigs in a system supplemented with both dihydropteridine reductase and cofactor. B y birth hydroxylase activity has reached adult levels. A comparison of the hydroxylase activity of adult rat liver and liver from a 23-26-week human fetus showed that the specific activity of the human fetal liver (0.043 moles tyrosine formed/rag protein/30 rain) is about 20 % that of the adult rat liver. To see if the hydroxylase present in h u m a n fetal liver is distinguishable from that of adult human liver by some of its kinetic propel"des, the apparent K~ values for phenylalanine and for dimethyltetrahydropterin were determined (Fig. 2a and b). The values of about 9 X 10-4M for phenylalanine and 4 X 10-~ M for dimethyltetrahydropterin are close to those obtained with adult human and adult rat liver phenylalanine hydroxylase [1 X 10-3 M for phenyIalanine and 5.7 N 10-~M for dimethyltetrahydropterin (24, 25)]. I t would appear from these data that in guinea pig and probably also in h u m a n fetal liver, phenylalanine hydroxylase development is similar to that in fetal chickens (13). I t is possible that in the rat phenylalanine hydroxylase is present in fetal liver, but in
an inhibited form. To examine this possibility an inactive extract from a 20-day fetus TABLE
III
PHI~NYLALANINE HYDROXYLASE ACTIVITIES FETAL: NEWBORN, AND ADULT GUINEA PIG
LIVER
IN
a
Tyrosine formed Age of animals
40-45-Day fetus ~ 45-50-Day fetus b 6-Hr newborns c Adult males
gmole/ml extract
umole/mg protein
0.257 0.675 2.393 2.320
0.013 0.031 0.098 0.089
The complete assay system is the same as the one described in Table I except for the source of extract. Dihydropteridine reductase (4.9 mg of protein) and dimethyltetrahydropterin (0.15 ~moles) are present in each experiment. b In eacn age group four fetal livers were pooled per experiment. c In this experiment two newborn livers were pooled.
PHENYLALANINE tIYDROXYLASE IN MAMMALS was incubated with an active extract from adult liver. There was no detectable inhibition. The possibility t h a t phenylalanine hydroxylase in 20-day rat fetal liver is a
//
40
2
325
particulate, rather t h a n a soluble, enzyme was excluded b y assays of hydroxylase activity in homogenates a n d in resuspended pellets derived from these homogenates; no activity was detected. A t t e m p t s to induce phenylalanine hydroxylase activity in 19-20-day fetuses by the injection of dihydrobiopterin intraperitoneally into the mother were unsuccessful; no hydroxylase activity was detected when the fetal livers were assayed 16-20 hr after the inieetion. D ISCUSSION
I I0
_,!2/_o!8 _o!4-o
04 o18
1.2
I/p HE NYLA L A N I N E (pmoles/rnl)
/
b
60
4C
20
-30
20
-I0 I/D ivl P H,~
~0 Lmoles/ml)
2'o ~o
Fro. 2. The effect of L-phenylalanine concentration and dimethyltetrahydropterin concentration on the rate of tyrosine formation catalyzed by fetal human liver phenylalanine hydroxylase. The assay system contained the following components (in micromoles unless otherwise stated): potassium phosphate buffer, pH 6.8, 100; TPNtt, 0.1; glucose-6-phosphate, 1; glucose-6-phosphate dehydrogenase, 5 ug; catMase, 100 ug; dihydropteridine reductase purified through the CaPO4 step (4), 0.1 rag. Volume was 1.0 nil, incubation time was 30 min, and the temperature was 25~ An extract (75 uliters), obtained from a homogenate made with 4 vol of 0.15 ~ KC1, was used in each assay. Tyrosine formation was determined fluorometrically by the nitrosonaphthol method (22). Velocity is expressed as umoles tyrosine formed in 30 min. a. Varying concentrations of L-phenylManine; dimethyltetrahydropterin 0.15 raM. b. Varying concentrations of dimethyltetrahydropterin; ~-phenylManine 2 m~. DMPH~ = dimethyltetrahydropterin.
While phenylalanine hydroxylase activity in g~inea pig and in h u m a n fetal liver is present much before term, it is only on the last d a y of fetal life t h a t the activity appears in rat fetal liver. The pattern of phenylalanine hydroxylase development in guinea pig and probably in h u m a n fetal liver, therefore, appears to be similar to that in fowl (13). I t is striking t h a t in all species studied, phenylalanine hydroxylase activity at birth is as high as in adults. I t is not clear why the rat, has such an unusual pattern of hydroxylase development,. We have a t t e m p t e d to demonstrate a proenzyme in inactive rat fetal liver extracts by looking for precipitation of the protein with antibody to highly purified adult rat liver phenylalanine hydroxylase, but these a t t e m p t s were unsueeessful (unpublished data). I t m a y be that a proenzyme is present but in a nonpreeipitable form, although we have no evidence to support this; our data are equally compatible with the contention t h a t all phenylalanine hydroxylase is synthesized during the last 24 hr of fetal life with the synthesis being induced b y an as yet undetermined factor. While adult h u m a n liver phenylalanine hydroxylase was not tested here, it has been shown previously that, the apparent K,~ values for phenylalanine and for 6,7-dimethyltetrahydropterin are very similar to those of the rat liver enzyme (25). Since we have now shown t h a t the K~ values for h u m a n fetal phenylala~fine hydroxylase are also very similar to those of the rat liver hydroxylase, we can conclude t h a t h u m a n fetal and adult phenylalanine hydroxylases
326
FRIEDMAN AND KAUFMAN
have similar K ~ values for phenylalanine and for the synthetic eofactor. The question of whether there exists a fetal form of phenylalanine hydroxylase in h u m a n liver cannot be answered unequivocally until more detailed kinetic studies are performed simultaneously on a t least partially purified enzyme from fetal and adult tissue or until a comparison of physical properties is made with highly purified enzyme from each tissue. Clinical studies have shown t h a t the heterozygote offspring of homozygous phenylketonuric women (not under dietary control) are usually mentally retarded and sometimes have associated congenital anomalies (26, 27). Thus, the development of phenylalanine hydroxylase activity during fetal life is not sufficient to prevent permanent neurological damage and tetratogenic effects which presumably result from a high fetal environment of phenylalanine. I t is of interest t h a t dihydropteridine reductase and the pterin cofactor are b o t h present at low, but significant, levels in fetal rat liver considerably earlier t h a n phenylalanine hydroxylase. Their presence at this time probably reflects their participation in some other hydroxylation reaction. On the other hand, there is a marked increase in the activities of both of these components of the phenylalanine-hydroxylating system on the last day of fetal life, at a time when the hydroxylase begins to appear. The stimulus to this seemingly coordinated increase in all components of the hydroxylating system is unknown. ACKNOWLEDGMENT The skillful technical assistance of Mr. Richard Funk is gratefully acknowledged. REFERENCES 1. KAUFMAN,S., J. Biol. Chem. 234, 2677 (1959). 2. KAUFMAN, S., Proc. Nat. Acad. Sci. U.S.A. 50, 1085 (1963).
3. KAUFMAN, S., AND LEYENBERG, B., J. Biol. Chem. 234, 2683 (1959). 4. KAUFMAN, S., or. Biol. Chem. 226, 511 (1957). 5. KENNEY, F. W., REEM, G. H., ANDKRETCHMER, N., Science 127, 86 (1958). 6. KENNEY~ F. T., AND KRETCHMER, J., J. Clin. Invest. 38, 2189 (1959). 7. NEMETH,A. M., J. Biol. Chem. 208,773 (1954).
8. NEMETH, A. M., AND NACHMIAS,V. T., Science
128, 1085 (1958). 9. WEBER, G., AND CANTERO, A., Cancer Res. 17, 995 (1957). 10. DAWKINS, M. J. R., Nature London 191, 72
(1961). 11. NEMETH,A. M., J. Biol. Chem. 234, 2921 (1959). 12. BRENNEMAN, A. R., AND KAUFMAN, S., J. Biol. Chem. 240, 3617 (1965). 13. STRITTMATER,C. E., AND OAKLEY, G., Proe. Soc. Exp. Biol. Med. 123, 427 (1966). 14. STRECKER, H. J., AND KORKES, S., J. Biol. Chem. 196, 769 (1952). 15. KAUFlVIAN,S., in "0xygenases" (O. Hayaishi, Ed.), p. 129. Academic Press, New York, 1962. 16. WALKER, D. G., ANn WIRTSCHAFTER, Z. T., in "The Genesis of the Rat Skeleton," p. 2. Thomas, Springfield, Ill., 1957. 17. KAUFMAN, S., J. Biol. Chem. 230, 931 (1958). 18. FISHER, D. B., AND KAUFMAN, S., Bioehem. Biophys. Res. Commun. 38, 663 (1970). 19. KATJFMAN, S., AND FISHER, D. B., J. Biol. Chem. 245, 4745 (1970). 20. SHIMAN, R., AKINO, M., AND KAUFMAN, S., J. Biol. Chem. 246, 1330 (1971).
21. UDENFRIEND, S., AND COOPER, J. R., J. Biol. Chem. 196, 227 (1952).
22. WAALKES, W. R., AND UDENFRIEND, S., J. Lab. Clin. Med. 50, 733 (1957). 23. LowRY, O. H., ROSENBOROUGH, N. J., FARR, h. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 24. KAUFMAN, S., Arch. Biochem. Biophys. 134, 249 (1969). 25. KAI~FMAN, S., Methods Enzymol. 17A, 597 (1970). 26. MABRY, C. C., DENNISTON, J. C., NELSON, W. L., AND SON, C. D., N. Eng. J. Med. 269, 1404 (1963). 27. Yu, J. S., AND O'~-]ALLORAN,M. T., Lancet 1, 210 (1970).
OF BIOCHEMISTRY
AND
BIOPHYSICS
146, 321-326 (1971)
A Study of the Development of Phenylalanine Hydroxylase in Fetuses of Several Mammalian Species P A U L A. F R I E D M A N AND S E Y M O U R K A U F M A N Laboratory of Neurochemistry, National Inslituge of Mental Heallh, Beghesda, Maryland 2001~ Received May 17, 1971; accepted June 11, 1971 The developmental pattern of hepatic phenylManine hydroxylase has been studied in several different mammals. Two strikingly different patterns have been observed. In the rat, no activity can be detected until the day before birth. The hydroxylase activity of newborn rats is equal to or slightly higher than that of adults. In the guinea pig and the human, in contrast, hydroxylase activity is detectable in the liver during the second third of the gestation period. I t is now well established that, the enzymatic conversion of phenylalanine to tyrosine in m a m m a l i a n liver is catalyzed b y a complex system consisting of several enzymes and eoenzymes. The minimum requirements are for phenylalanine hydroxylase and a tetrahydropterin eofaetor (1). The eofaetor requirement can be filled by either the naturally occurring compound, tetrahydrobiopterin (2), or b y one of several synthetic analogs, such as 6,7-dimethyltetrahydropterin (3): In the presence of catalytic concentrations of the pterin cofactor, the hydroxylation reaction is also dependent on a tetrahydropterin-regenerating system, such as dihydropteridine reductase and T P N H (4). I n 1958 (5, 6), it, was reported t h a t phenylalanine hydroxylase was a m e m b e r of a group of m a m m a l i a n liver enzymes t h a t are essentially inactive at birth (7-11). The activities of these enzymes increase rapidly after birth, usually reaching the adult levels within 1 or 2 days (7-11). I n 1965, we showed t h a t phenylalanine hydroxylase did not belong to this group of enzymes t h a t are inactive at birth. I t was found t h a t when properly supplemented with dihydropteridine reductase and pterin cofaetor, the hydroxylase activity in livers from newborn rats is equal to, or higher than, t h a t of adults (12). The levels of the pterin
cofactor and of dihydropteridine reductase in the newborn livers were relatively deficient (12). A study of the development of phenylalanine hydroxylase activity in fetal chicken liver showed t h a t the activity appeared during the second half of the incubation period and approached adult levels at t e r m (13). I n the present paper, we report the results of studies on the deve]opmentM pattern of phenylalanine hydroxylase in the fetal livers of several m a m m a l i a n species. Two strikingly different patterns have been observed. I n the guinea pig and the human, hydroxylase activity is detectable in fetal livers during the second third of the gestation period. B y contrast, in the rat, no activity is detected until the day before birth. MATERIALS AND METHODS
Sprague-Dawley rats and Hartley guinea pigs were obtained from the National Institutes of Health animal supply section. Liver tissue from a 23-week-old human fetus was kindly supplied by Dr. Neil Holtzman of Johns Hopkins Hospital, Baltimore, Md. The liver was removed from the fetus within 1 hr of a therapeutic abortion and was quick frozen and stored at -80 ~. The activity was measured after 4 weeks of frozen storage. Tyrosine and phenylalanine were obtained from NutritionM Biochemicals. TPN + was purchased from Sigma Chemical Co., and glucose-6321
322
FRIEDMAN AND KAUFMAN
phosphate from Calbioehem. Catalase, glucose-6phosphate-dehydrogenase, and TPNH were obtained from Boehringer Mannheim. 2-Amino-4hydroxy - 6,7 - dimethyl - 5,6,7,8 - tetrahydropteridine was purchased from Aldrich Chemical Co. Glucose dehydrogenase was prepared by a modification of the method by Strecker and Korkes (14); the fractions used supply adequate amounts of catalase, an enzyme that is required to protect the hydroxylating system against tI20~-mediated inactivation (15). Preparation of tissues. Fetal animMs were delivered by Cesarean section immediately after the mothers had been killed by cervical fracture. Guinea pig fetal age was approximated from fetal size and estimated date of conception. Rat fetal age was estimated from the degree of skeletal development with the use of a modification of the clearing and staining procedures of Walker and Wirtschrafter (16). Fetal and newborn animals were killed by decapitation; adult animals by cervical fracture. All steps were performed at 0-4 ~ Livers obtained from fetal or newborn animals were pooled for experiments. They were cooled and washed in 0.25M sucrose, blotted, weighed, minced, and homogenized manually in an all-glass PotterElvehjem homogenizer. Extracts for hydroxylase assays were prepared by homogenization of the livers with 3 vol of 0.15 M KC1 unless otherwise stated. When human fetal liver was to be assayed, a small, still frozen portion of tissue was removed from the bulk of the liver with a razor blade in a cold room at - 2 0 ~ Prior to homogenization, the required volume of XC1 solution was added to the frozen tissue which was then allowed to thaw at 4 ~ The homogenates were centrifuged at 16,000g for 15 min, and the supernatant fluids were immediately used to assay for phenylalanine hydroxylase or dihydropteridine reductase activities. For cofactor assays, the liver extracts were prepared as described above except that 2 vol of glass-distilled water was used for homogenization. The supernatant fluids were boiled for 2 min with stirring. They were rapidly cooled and centrifuged for 15 rain at 16,00~g. The clear yellow supernatant fluid was assayed for phenylManine hydroxylation cofactor activity (17). Enzyme assays. Phenylalanine hydroxylase was assayed according to published procedures (4, 18); the incubation period was either 30 or 45 rain. Dihydropteridine reductase was assayed by a modification of the standard phenylalanine hydroxylase assay system (4). Excess amounts of phenylalanine hydroxylase prepared from rat liver and purified through the Sephadex G-200
step were used (19). Synthetic dimethyltetrahydropterin (1) was used as the cofactor at a concentration of 0.3 mM. In all experiments, the concentration of the pterin was calculated from a measurement of its absorbance in 2 N perchlorie acid (extinction coefficient = 17.8 X 10s ~-i cm-1 at 265 mt~) (20). Tyrosine formation was measured either speetrophotometrically or fluorometricMly by the nitrosonaphthol method (21, 22). Protein was determined by the Lowry method with the use of bovine serum albumin as the standard (23). RESULTS T a b l e I shows a c o m p a r i s o n of tile act i v i t y of the p h e n y l a l a n i n e - h y d r o x y l a t i n g s y s t e m i n livers from fetal, n e w b o r n , a n d a d u l t male rats u n d e r v a r i o u s conditions. As f o u n d previously (12), w i t h o u t supplem e n t a t i o n , n e w b o r n livers h a v e significant a c t i v i t y , a l t h o u g h lower t h a n t h a t of a d u l t males. 1 T o d e t e r m i n e w h e t h e r the r e l a t i v e l y low a c t i v i t y of t h e n e w b o r n s was due to a deficiency of d i h y d r o p t e r i d i n e reductase, cofactor, or p h e n y l a l a n i n e hydroxylase, t h e s y s t e m was assayed after s u p p l e m e n t a t i o n with d i h y d r o p t e r i d i n e reductase a n d / o r cofactor. T h e results, s h o w n i n T a b l e I, are i n a g r e e m e n t with those previously r e p o r t e d (12). I n a s y s t e m s u p p l e m e n t e d with both d i h y d r o p t e r i d i n e r e d u c t a s e a n d cofactor the h y d r o x y l a s e a c t i v i t y i n n e w b o r n livers is at least equal to t h a t of adults. T h e greater s t i m u l a t i o n of t h e n e w b o r n liver extracts b y cofactor a n d d i h y d r o p t e r i d i n e r e d u c t a s e is i n a g r e e m e n t with previous o b s e r v a t i o n s (12) a n d supports the conclusion t h a t b o t h of these c o m p o n e n t s are relatively deficient i n the n e w b o r n livers (12). Similar studies with fetal livers ( T a b l e I) d e m o n s t r a t e t h a t i n t h e system supplem e n t e d with b o t h d i h y d r o p t e r i d i n e reductase a n d cofactor, no significant hydroxylase a c t i v i t y is detectable earlier t h a n 24 to 36 The hydroxylase activities of both the newborn and adult liver extracts are about 60% higher than those reported previously (12). The only known variables between the previous and present procedures are: (a) gentle hand homogenization of the livers was used in the present procedure instead of the previously used motor~driven homogelfizer, and (b) somewhat higher levels of the pterin cofactor were used in the present assays (0.30 m~ versus 0.22 m~).
323
P H E N Y L A L A N I N E H Y D R O X Y L A S E IN M A M M A L S TABLE I 1)ItENYLALANINE HYDROXYLASE
NEWBORN~
A C T I V I T I E S IN F E T A L ,
AND
ADULT
t~ATS a
Tyrosine formed Age of animalsb
No additions ~mole/ml extract
D a y 14-17 fetus Late d a y 18 fetus ]Day 21 fetus E a r l y d a y 22 fetus L a t e d a y 22 fetus L a t e d a y 22 featus (during active labor) 6-Hr n e w b o r n A d u l t males~
q- Dihydropteridine reductase
q- Dihydropteridine reductase and tetrahydropterin
-~ Tetrahydropterin
gmole/mg ~mole/ml /*mole/rag t~mole/ml amole/mg ~mole/ml ~ole/mg extract protein protein protein extract protein extract
0 0 0 0.018 0.093
0 0 0 0. 002 0.008
0 0 0 0. 045 0.113
0 0 0 0. 004 0.010
0 0 0 0.225 1.161
0 0 0 0.019 0.106
0 0 0 0.263 1.610 2.101
0 0 0 0.022 0.146 0.168
0.210 0.580 4-0.040
0.013 0.027 4-0.004
0.240 0.704 4-0.040
0.015 0.033 -t-0.003
1.143 1.452
0.072 0.068 4-0.003
2.613 2.145
0.157 0.099
•
•
•
T h e complete s y s t e m c o n t a i n e d t h e following components (in micromoles unless otherwise s t a t e d ) : p o t a s s i u m p h o s p h a t e buffer, p H 6.8, 50: L-phenylManine, 1.0; T P N +, 0.15; glucose, 125; glucose dehydrogenase, 75 milts; liver e x t r a c t , 0.2 ml. D i h y d r o p t e r i d i n e r e d u c t a s e (4.9 mg of p r o t e i n ) and dim e t h y l t e t r a h y d r o p t e r i n (0.15 ~mole) were added where indicated. T h e final volume was 0.5 nil, t h e i n c u b a t i o n t i m e was 45 min, and t h e t e m p e r a t u r e was 25 ~ F r o m each g r a m wet w e i g h t of liver 2.6 ml of e x t r a c t is obtaineff. b E a c h experiment w i t h fetal or n e w b o r n animals represents 6-12 pooled livers per litter. Results are an average of four experiments 4- s t a n d a r d deviation.
hr prior to parturition. There follows a dramatic rise in activity (Fig. 1) until late in l'~bor hydroxylase activity reaches adult levels. If fetal liver is supplemented with either dihydropteridine reductase or with cofactor, there is stimulation of hydroxylase activity. The stimulation with cofactor is much more striking and accounts for much of the effect seen in the system supplemented with both dihydropteridine reductase and cofactor. These data suggest that in fetal livers which show hydroxylase activity, cofactor levels are deficient when compared to livers of newborns and adults. The possibility that all three essential components of the phenylalanine-hydroxylating system develop together was examined by assaying livers of several fetuses of different ages for dihydropteridine reductase and for cofactor activity (Table II). It is apparent that in fetal livers which have no hydroxylase activity (day 18, day 20-21), both dihydropteridine reductase and cofactor are present. In the last 24-48 hr of fetal life, however, there is more than a
5.0 E --L i
2.0'
\
~o
0 ':t
~- 0
16 18 GESTATION AGE (Days)
FIG. 1. P h e n y l a l a n i n e hydroxylase activity in fetal, newborn, a n d adult r a t liver. This curve is a plot of the d a t a in T a b l e I in t h e system to which d i h y d r o p t e r i d i n e reductase and d i m e t h y l t e t r a h y d r o p t e r i n have been added. N = 6-hr newborn; A = adult.
twofold increase in both activities. Thus, although the appearance in fetal life of the three components of the phenyla]aninehydroxylating system is not simultaneous,
324
FRIEDMAN AND KAUFMAN TABLE II
DIHYDROPTERIDINE I:~EDUCTASE AND PHENYL ~ ALANINE ]-IyDROXYLATIO N COFACTOR ACTIVITIES IN FETAL AND ADULT I~ATSa
Experimental animals
Dihydropteridlne reductase activity (tyrosine formed) ttmole/ml extract
Day-18 fetus Day-20-21 fetus Late day-22 fetus
(during labor) Adult males b
0.115 0.135 0.300
#mole/rag protein
0.012 0.014 0.024
Cofactor activity (uaits/ml extract)
0.056 0.065 0.169
0.885 0.048 0. 532 4-0.042 =t=0.004 =t=0.043
The complete assay system for dihydropteridine reductase activity is described under Materials and Methods. The complete assay system for cofactor activity contained the stone components as described in Table I with the following exceptions: PhenylManine hydroxylase from rats (0.180 mg of protein), purified through the G-200 Sephadex step (19) and dihydropteridine reduetase from sheep (9.8 mg of protein), purified through the first ammonium sulfate precipitation (4), were used. Cofactor activity was determined on boiled extracts of liver (0.4 ml) prepared as described in Materials and Methods. In both assays, the volume was 1.0 ml and incubations were for 45 min at 25~ One unit of eofaetor activity represents the additional formation of 1.0 t,moles of tyrosine under the standard assay conditions (17). b Average of two experiments 4- range. there is a similar marked increase in the activities of phenylalanine hydroxylase, dihydropteridine reduetase, and eofaetor just prior to birth. The development of phenylalanine hydroxylase activity in fetal rats is quite different from that reported for fetal chickens (13). I n chickens, enzyme activity appears at about fetal day 12 of a 20-day incubation period and reaches approximately adult levels b y fetal day 17. To determine if the pattern in fetal rats is representative of the development of phenylalanine hydroxylase in mammals in general, guinea pig and human fetal liver were also studied. Table I I I shows that liver from a 40-45day fetal guinea pig (gestation period 64 days) already has one-tenth of the activity
of adult male guinea pigs in a system supplemented with both dihydropteridine reductase and cofactor. B y birth hydroxylase activity has reached adult levels. A comparison of the hydroxylase activity of adult rat liver and liver from a 23-26-week human fetus showed that the specific activity of the human fetal liver (0.043 moles tyrosine formed/rag protein/30 rain) is about 20 % that of the adult rat liver. To see if the hydroxylase present in h u m a n fetal liver is distinguishable from that of adult human liver by some of its kinetic propel"des, the apparent K~ values for phenylalanine and for dimethyltetrahydropterin were determined (Fig. 2a and b). The values of about 9 X 10-4M for phenylalanine and 4 X 10-~ M for dimethyltetrahydropterin are close to those obtained with adult human and adult rat liver phenylalanine hydroxylase [1 X 10-3 M for phenyIalanine and 5.7 N 10-~M for dimethyltetrahydropterin (24, 25)]. I t would appear from these data that in guinea pig and probably also in h u m a n fetal liver, phenylalanine hydroxylase development is similar to that in fetal chickens (13). I t is possible that in the rat phenylalanine hydroxylase is present in fetal liver, but in
an inhibited form. To examine this possibility an inactive extract from a 20-day fetus TABLE
III
PHI~NYLALANINE HYDROXYLASE ACTIVITIES FETAL: NEWBORN, AND ADULT GUINEA PIG
LIVER
IN
a
Tyrosine formed Age of animals
40-45-Day fetus ~ 45-50-Day fetus b 6-Hr newborns c Adult males
gmole/ml extract
umole/mg protein
0.257 0.675 2.393 2.320
0.013 0.031 0.098 0.089
The complete assay system is the same as the one described in Table I except for the source of extract. Dihydropteridine reductase (4.9 mg of protein) and dimethyltetrahydropterin (0.15 ~moles) are present in each experiment. b In eacn age group four fetal livers were pooled per experiment. c In this experiment two newborn livers were pooled.
PHENYLALANINE tIYDROXYLASE IN MAMMALS was incubated with an active extract from adult liver. There was no detectable inhibition. The possibility t h a t phenylalanine hydroxylase in 20-day rat fetal liver is a
//
40
2
325
particulate, rather t h a n a soluble, enzyme was excluded b y assays of hydroxylase activity in homogenates a n d in resuspended pellets derived from these homogenates; no activity was detected. A t t e m p t s to induce phenylalanine hydroxylase activity in 19-20-day fetuses by the injection of dihydrobiopterin intraperitoneally into the mother were unsuccessful; no hydroxylase activity was detected when the fetal livers were assayed 16-20 hr after the inieetion. D ISCUSSION
I I0
_,!2/_o!8 _o!4-o
04 o18
1.2
I/p HE NYLA L A N I N E (pmoles/rnl)
/
b
60
4C
20
-30
20
-I0 I/D ivl P H,~
~0 Lmoles/ml)
2'o ~o
Fro. 2. The effect of L-phenylalanine concentration and dimethyltetrahydropterin concentration on the rate of tyrosine formation catalyzed by fetal human liver phenylalanine hydroxylase. The assay system contained the following components (in micromoles unless otherwise stated): potassium phosphate buffer, pH 6.8, 100; TPNtt, 0.1; glucose-6-phosphate, 1; glucose-6-phosphate dehydrogenase, 5 ug; catMase, 100 ug; dihydropteridine reductase purified through the CaPO4 step (4), 0.1 rag. Volume was 1.0 nil, incubation time was 30 min, and the temperature was 25~ An extract (75 uliters), obtained from a homogenate made with 4 vol of 0.15 ~ KC1, was used in each assay. Tyrosine formation was determined fluorometrically by the nitrosonaphthol method (22). Velocity is expressed as umoles tyrosine formed in 30 min. a. Varying concentrations of L-phenylManine; dimethyltetrahydropterin 0.15 raM. b. Varying concentrations of dimethyltetrahydropterin; ~-phenylManine 2 m~. DMPH~ = dimethyltetrahydropterin.
While phenylalanine hydroxylase activity in g~inea pig and in h u m a n fetal liver is present much before term, it is only on the last d a y of fetal life t h a t the activity appears in rat fetal liver. The pattern of phenylalanine hydroxylase development in guinea pig and probably in h u m a n fetal liver, therefore, appears to be similar to that in fowl (13). I t is striking t h a t in all species studied, phenylalanine hydroxylase activity at birth is as high as in adults. I t is not clear why the rat, has such an unusual pattern of hydroxylase development,. We have a t t e m p t e d to demonstrate a proenzyme in inactive rat fetal liver extracts by looking for precipitation of the protein with antibody to highly purified adult rat liver phenylalanine hydroxylase, but these a t t e m p t s were unsueeessful (unpublished data). I t m a y be that a proenzyme is present but in a nonpreeipitable form, although we have no evidence to support this; our data are equally compatible with the contention t h a t all phenylalanine hydroxylase is synthesized during the last 24 hr of fetal life with the synthesis being induced b y an as yet undetermined factor. While adult h u m a n liver phenylalanine hydroxylase was not tested here, it has been shown previously that, the apparent K,~ values for phenylalanine and for 6,7-dimethyltetrahydropterin are very similar to those of the rat liver enzyme (25). Since we have now shown t h a t the K~ values for h u m a n fetal phenylala~fine hydroxylase are also very similar to those of the rat liver hydroxylase, we can conclude t h a t h u m a n fetal and adult phenylalanine hydroxylases
326
FRIEDMAN AND KAUFMAN
have similar K ~ values for phenylalanine and for the synthetic eofactor. The question of whether there exists a fetal form of phenylalanine hydroxylase in h u m a n liver cannot be answered unequivocally until more detailed kinetic studies are performed simultaneously on a t least partially purified enzyme from fetal and adult tissue or until a comparison of physical properties is made with highly purified enzyme from each tissue. Clinical studies have shown t h a t the heterozygote offspring of homozygous phenylketonuric women (not under dietary control) are usually mentally retarded and sometimes have associated congenital anomalies (26, 27). Thus, the development of phenylalanine hydroxylase activity during fetal life is not sufficient to prevent permanent neurological damage and tetratogenic effects which presumably result from a high fetal environment of phenylalanine. I t is of interest t h a t dihydropteridine reductase and the pterin cofactor are b o t h present at low, but significant, levels in fetal rat liver considerably earlier t h a n phenylalanine hydroxylase. Their presence at this time probably reflects their participation in some other hydroxylation reaction. On the other hand, there is a marked increase in the activities of both of these components of the phenylalanine-hydroxylating system on the last day of fetal life, at a time when the hydroxylase begins to appear. The stimulus to this seemingly coordinated increase in all components of the hydroxylating system is unknown. ACKNOWLEDGMENT The skillful technical assistance of Mr. Richard Funk is gratefully acknowledged. REFERENCES 1. KAUFMAN,S., J. Biol. Chem. 234, 2677 (1959). 2. KAUFMAN, S., Proc. Nat. Acad. Sci. U.S.A. 50, 1085 (1963).
3. KAUFMAN, S., AND LEYENBERG, B., J. Biol. Chem. 234, 2683 (1959). 4. KAUFMAN, S., or. Biol. Chem. 226, 511 (1957). 5. KENNEY, F. W., REEM, G. H., ANDKRETCHMER, N., Science 127, 86 (1958). 6. KENNEY~ F. T., AND KRETCHMER, J., J. Clin. Invest. 38, 2189 (1959). 7. NEMETH,A. M., J. Biol. Chem. 208,773 (1954).
8. NEMETH, A. M., AND NACHMIAS,V. T., Science
128, 1085 (1958). 9. WEBER, G., AND CANTERO, A., Cancer Res. 17, 995 (1957). 10. DAWKINS, M. J. R., Nature London 191, 72
(1961). 11. NEMETH,A. M., J. Biol. Chem. 234, 2921 (1959). 12. BRENNEMAN, A. R., AND KAUFMAN, S., J. Biol. Chem. 240, 3617 (1965). 13. STRITTMATER,C. E., AND OAKLEY, G., Proe. Soc. Exp. Biol. Med. 123, 427 (1966). 14. STRECKER, H. J., AND KORKES, S., J. Biol. Chem. 196, 769 (1952). 15. KAUFlVIAN,S., in "0xygenases" (O. Hayaishi, Ed.), p. 129. Academic Press, New York, 1962. 16. WALKER, D. G., ANn WIRTSCHAFTER, Z. T., in "The Genesis of the Rat Skeleton," p. 2. Thomas, Springfield, Ill., 1957. 17. KAUFMAN, S., J. Biol. Chem. 230, 931 (1958). 18. FISHER, D. B., AND KAUFMAN, S., Bioehem. Biophys. Res. Commun. 38, 663 (1970). 19. KATJFMAN, S., AND FISHER, D. B., J. Biol. Chem. 245, 4745 (1970). 20. SHIMAN, R., AKINO, M., AND KAUFMAN, S., J. Biol. Chem. 246, 1330 (1971).
21. UDENFRIEND, S., AND COOPER, J. R., J. Biol. Chem. 196, 227 (1952).
22. WAALKES, W. R., AND UDENFRIEND, S., J. Lab. Clin. Med. 50, 733 (1957). 23. LowRY, O. H., ROSENBOROUGH, N. J., FARR, h. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 24. KAUFMAN, S., Arch. Biochem. Biophys. 134, 249 (1969). 25. KAI~FMAN, S., Methods Enzymol. 17A, 597 (1970). 26. MABRY, C. C., DENNISTON, J. C., NELSON, W. L., AND SON, C. D., N. Eng. J. Med. 269, 1404 (1963). 27. Yu, J. S., AND O'~-]ALLORAN,M. T., Lancet 1, 210 (1970).