682
Biochimica et Biophysica Acta, 4 9 7 ( 1 9 7 7 ) 6 8 2 - - 6 8 9 © Elsevier/North-Holland Biomedical Press
BBA 28234
PHENYLALANINE-PYRUVATE AMINOTRANSFERASE IN IMMATURE AND ADULT MAMMALIAN TISSUES INDUCTION IN FETAL RAT LIVER *
LETICIA SANCHEZ-URRETIA
** a n d O L G A G R E E N G A R D
Department o f Biological Chemistry, Harvard Medical School, and the Cancer Research Institute, New England Deaconess Hospital, Boston, Mass. 02215 (U.S.A.) (Received November 8th, 1976)
Summary Normal human fetal liver contains little phenylalanine-pyruvate aminotransferase: between the l l t h and 22nd week of gestation its activity (per g) is 8.8% of that in adult liver. In rat liver this enzyme begins to rise a few hours before birth. Precocious increases in the phenylalanine-pyruvate aminotransferase activity of fetal rat liver (but n o t kidney or brain) were evoked by premature delivery and also by the administration of thyroxine or glucagon in utero. These results, discussed in relation to related observations on other enzymes, suggest that thyroxine secreted by the fetus, and also another factor released at the beginning of labour, may be the natural stimuli for the developmental formation of phenylalanine-pyruvate aminotransferase. The regulation of hepatic phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) during fetal development is different: in both man and rat, phenylalanine hydroxylase begins to rise earlier and is unaffected by the treatments which enhanced the formation of phenylalanine-pyruvate aminotransferase. In suckling rats (but not in fetuses and adults), an injection of cortisol increased the levels of both enzymes. Hepatocarcinomas of the adult rat were devoid of phenylalanine hydroxylase as well as phenylalanine-pyruvate aminotransferase. However, suppression in vivo by substrate analogues (a-methylphenylalanine and p-chlorophenylalanine) was unique for phenylalanine hydroxylase.
* This is article 580 from the Cancer Research Institute of the New England Deaconess Hospital. ** Present address: D e p a r t m e n t of Biochemistry and Molecular Biology, Faculty of Sciences, Auton o m o u s University of Madrid, Spain.
683 Introduction Investigations, in this laboratory, of biochemical differentiation included several studies of rat liver enzymes involved in the metabolism of aromatic amino acids [1--3]. Two of these, tyrosine aminotransferase and phenylalanine hydroxylase, have also been quantified in immature and adult human liver [4]. Recent attempts to create rat models for the human disease of phenylketonuria led to the identification of a-methylphenylalanine as an effective inducer of hyperphenylalaninemia [ 5 ] and to some new observations about the regulation of the hepatic phenylalanine hydroxylase in vivo [6]. Phenylalanine-pyruvate aminotransferase, the other important catalyst of phenylalanine metabolism, has received relatively little attention. However, an early report [7] did show that in rat liver its concentration is low during gestation, and later studies [8,9] described its induction in adult rat liver by glucagon. The age at which phenylalanine-pyruvate aminotransferase appears in human liver, or the stimuli responsible for its developmental formation in rat liver, have n o t been investigated. This was the main aim of the present studies. It was also of interest to test if phenylalanine-pyruvate aminotransferase responds to any of the positive or negative regulators of phenylalanine hydroxylase, and whether or not it undergoes adaptive changes during acute or chronic hyperphenylalaninemia. The results show that, in human liver at midgestation, phenylalanine-pyruvate aminotransferase is still at negligible levels. In fetal rat liver, precocious rises in phenylalanine-pyruvate aminotransferase could be evoked with an injection of glucagon or thyroxine or by premature delivery. Mechanisms involved in stimulating the developmental synthesis of hepatic enzymes, and differences between the regulation of phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase, will be discussed. Materials and Methods Tissues of fetuses from cases of elective abortions were kindly provided by Dr. Shirley Driscoll of the Boston Hospital for Women. The human adult liver and lung specimens were portions of surgical biopsy samples, which appeared to be normal upon histological examination. For additional details of studies on these human tissues see Herzfeld et al. [10] and DelValle and Greengard [4]. Normal rats were of the albino CDF strain, except those (Buffalo rats) which carried the transplantable Morris hepatoma 7777. Adult rats were 6 0 - 9 0 - d a y old males. There was an approximate 12 h error in the estimation of the fetal ages; they were based on time mating and b o d y weight [ 11 ]. In prenatal experiments, the anesthetized dams were laparotomized and fetuses were given intraperitoneal injections of hormones (alternate fetuses received vehicle only) through the uterine walls. The maternal abdomen was then closed; fetal death during the subsequent 24 h was very rare. Substances to be injected were dissolved or suspended in 0.9% NaC1; controls received the same volume of the vehicle only. The doses of hydrocortisone acetate (Merck Sharp and Dohme, Co., West Point, Pa, U.S.A.), glucagon (Eli Lilly and Co., Indianapolis, Ind., U.S.A.), thyroxine (Calbiochem, San Diego, Calif.,
684 U.S.A.) and epinephrine (Parke, Davis and Co., Detroit, Mich., U.S.A.) were 0.125, 0.003, 0.05, and 0.01 mg per fetus, respectively. Postnatal rats received intraperitoneal injections of hydrocortisone acetate or thyroxine, 2.5 and 0.003 mg per 100 g body weight, respectively. The preparations of L-phenylalanine, p-chlorophenylalanine and a-methylphenylalanine suspensions to be injected were described in a preceding paper [121. Rats were killed by decapitation. Freshly excised tissues were homogenized in four voumes of chilled 0.25 M sucrose and centrifuged at 105 000 × g for 1 h. The supernatant fluids were used for the enzyme assay. The human tissues (as described previously [4]) were processed in the same way. Phenylalanine-pyruvate aminotransferase was assayed at 30°C by the method of Spolter and Baldridge [13]. Reaction mixtures of 3.4 ml final volume contained 0.2 ml of tissue extract, sodium borate (0.58 M)/sodium arsenate (19 mM) buffer, pH 8.5, phenylalanine (58.7 raM) pyridoxal 5-phosphate (5 pg), EDTA (23.5 raM), pH 8.5, and pyruvate (44.1 mM), pH 8.5. The absorbance changes were followed in a Gilford recording spectrophotometer. Phenylalanine hydroxylase was assayed as described by McGee et al. [3] at 25°C. Enzyme activities are expressed in units (pmol per min) per g wet weight. Results The distribution of phenylalanine-pyruvate aminotransferase among some human and rat tissues is presented in Table I. The fetal human liver contained no appreciable activity; in the fetal rat the hepatic activity was 6.5 times lower than in the adult. None could be detected in the lungs {fetal or adult) of either species. The activity in rat brain was significant, about one-sixth of that in adult rat liver, and remained constant throughout development (see Fig. 1). Of the two abnormal tissues studied, rat hepatoma exhibited no phenylalaninepyruvate aminotransferase activity, and the liver of the tumor-bearing host contained about half as much as did that of normal rats of the same age. For comparison, the last two columns of Table I list some observations on phenylalanine hydroxylase. It may be seen that at the same fetal period phenylalanine hydroxylase has attained a much higher concentration (57% of that in the adult) than did phenylalanine-pyruvate aminotransferase. Another difference between the two enzymes is that only phenylalanine-pyruvate aminotransferase decreased in tumor-bearing rats; however, both enzymes were absent from the hepatoma itself. Sequential changes with age in the phenylalanine-pyruvate aminotransferase content of rat tissues are shown in Fig. 1. In kidney most of the increase occurred during postnatal life. The observations on liver differed only in detail from. those of Auerbach and Waisman [7]. We noted a multiphasic pattern. The most rapid increase occurred during the few hours before birth; this was followed by a partial loss and a second, slow phase of rise t o o k place between the 5th and 16th postnatal days. Experiments designed to identify physiological stimuli which may be responsible for the developmental formation of hepatic phenylalanine-pyruvate
685 TABLE I THE DISTRIBUTION OF PHENYLALANINE-PYRUVATE AND ADULT TISSUES OF MAN AND RAT
AMINOTRANSFERASE
AMONG
FETAL
T h e v a l u e s r e f e r t o i n d i v i d u a l s o r axe m e a n s +- S.D. o f r e s u l t s w i t h t h e n u m b e r o f t i s s u e s i n p a r e n t h e s e s . P h e n y l a l a n i n e - p y r u v a t e a m i n o t r a n s f e r a s e was m e a s u r e d in the liver a n d l u n g of 1 8 - - 2 0 w e e k h u m a n fetuses. The adult and fetal (11th--22nd week of gestation) values for human hepatic phenylalanine h y d r o x y l a s e are f r o m a p r e v i o u s p u b l i c a t i o n [ 4 ] . T h e s o l u b l e p r o t e i n c o n t e n t o f h u m a n a d u l t a n d f e t a l l i v e r w a s 1 0 2 ÷ 1 1 ( 3 ) a n d 6 8 +- 4 ( 6 ) , r e s p e c t i v e l y . T h e c o n c e p t u a l age o f r a t f e t u s e s w a s 2 0 d a y s . H o s t livers were from rats 30--45 days after the transplantation, when the weight of the hepatomas were 60-9 0 g ; t h e e n z y m e a c t i v i t i e s i n n o r m a l a d u l t l i v e r s o f t h e s e B u f f a l o r a t s w e r e t h e s a m e as t h o s e o f t h e s t r a i n u s e d i n all t h e o t h e r e x p e r i m e n t s . Tissue
Age
Enzyme activity (units/g) Phenylalanine-py ruvat e aminotransferase
Phenylalanine hydroxylase
Man
Rat
Man
0.57, 0.32 0 . 0 4 +- 0 . 0 3 (4)
1 . 3 +- 0 . 1 (9) 0 . 2 0 +- 0 . 0 2 ( 4 )
0 . 3 1 +- 0 . 0 7 ( 5 ) 0 . 1 8 -+ 0 . 0 6 ( 6 )
Liver
Adult Fetal
Lung
Adult Fetal
Brain
Adult Fetal
---
Hepatoma
Adult
--
Host liver
Adult
--
0.07
Biochimica et Biophysica Acta, 4 9 7 ( 1 9 7 7 ) 6 8 2 - - 6 8 9 © Elsevier/North-Holland Biomedical Press
BBA 28234
PHENYLALANINE-PYRUVATE AMINOTRANSFERASE IN IMMATURE AND ADULT MAMMALIAN TISSUES INDUCTION IN FETAL RAT LIVER *
LETICIA SANCHEZ-URRETIA
** a n d O L G A G R E E N G A R D
Department o f Biological Chemistry, Harvard Medical School, and the Cancer Research Institute, New England Deaconess Hospital, Boston, Mass. 02215 (U.S.A.) (Received November 8th, 1976)
Summary Normal human fetal liver contains little phenylalanine-pyruvate aminotransferase: between the l l t h and 22nd week of gestation its activity (per g) is 8.8% of that in adult liver. In rat liver this enzyme begins to rise a few hours before birth. Precocious increases in the phenylalanine-pyruvate aminotransferase activity of fetal rat liver (but n o t kidney or brain) were evoked by premature delivery and also by the administration of thyroxine or glucagon in utero. These results, discussed in relation to related observations on other enzymes, suggest that thyroxine secreted by the fetus, and also another factor released at the beginning of labour, may be the natural stimuli for the developmental formation of phenylalanine-pyruvate aminotransferase. The regulation of hepatic phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) during fetal development is different: in both man and rat, phenylalanine hydroxylase begins to rise earlier and is unaffected by the treatments which enhanced the formation of phenylalanine-pyruvate aminotransferase. In suckling rats (but not in fetuses and adults), an injection of cortisol increased the levels of both enzymes. Hepatocarcinomas of the adult rat were devoid of phenylalanine hydroxylase as well as phenylalanine-pyruvate aminotransferase. However, suppression in vivo by substrate analogues (a-methylphenylalanine and p-chlorophenylalanine) was unique for phenylalanine hydroxylase.
* This is article 580 from the Cancer Research Institute of the New England Deaconess Hospital. ** Present address: D e p a r t m e n t of Biochemistry and Molecular Biology, Faculty of Sciences, Auton o m o u s University of Madrid, Spain.
683 Introduction Investigations, in this laboratory, of biochemical differentiation included several studies of rat liver enzymes involved in the metabolism of aromatic amino acids [1--3]. Two of these, tyrosine aminotransferase and phenylalanine hydroxylase, have also been quantified in immature and adult human liver [4]. Recent attempts to create rat models for the human disease of phenylketonuria led to the identification of a-methylphenylalanine as an effective inducer of hyperphenylalaninemia [ 5 ] and to some new observations about the regulation of the hepatic phenylalanine hydroxylase in vivo [6]. Phenylalanine-pyruvate aminotransferase, the other important catalyst of phenylalanine metabolism, has received relatively little attention. However, an early report [7] did show that in rat liver its concentration is low during gestation, and later studies [8,9] described its induction in adult rat liver by glucagon. The age at which phenylalanine-pyruvate aminotransferase appears in human liver, or the stimuli responsible for its developmental formation in rat liver, have n o t been investigated. This was the main aim of the present studies. It was also of interest to test if phenylalanine-pyruvate aminotransferase responds to any of the positive or negative regulators of phenylalanine hydroxylase, and whether or not it undergoes adaptive changes during acute or chronic hyperphenylalaninemia. The results show that, in human liver at midgestation, phenylalanine-pyruvate aminotransferase is still at negligible levels. In fetal rat liver, precocious rises in phenylalanine-pyruvate aminotransferase could be evoked with an injection of glucagon or thyroxine or by premature delivery. Mechanisms involved in stimulating the developmental synthesis of hepatic enzymes, and differences between the regulation of phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase, will be discussed. Materials and Methods Tissues of fetuses from cases of elective abortions were kindly provided by Dr. Shirley Driscoll of the Boston Hospital for Women. The human adult liver and lung specimens were portions of surgical biopsy samples, which appeared to be normal upon histological examination. For additional details of studies on these human tissues see Herzfeld et al. [10] and DelValle and Greengard [4]. Normal rats were of the albino CDF strain, except those (Buffalo rats) which carried the transplantable Morris hepatoma 7777. Adult rats were 6 0 - 9 0 - d a y old males. There was an approximate 12 h error in the estimation of the fetal ages; they were based on time mating and b o d y weight [ 11 ]. In prenatal experiments, the anesthetized dams were laparotomized and fetuses were given intraperitoneal injections of hormones (alternate fetuses received vehicle only) through the uterine walls. The maternal abdomen was then closed; fetal death during the subsequent 24 h was very rare. Substances to be injected were dissolved or suspended in 0.9% NaC1; controls received the same volume of the vehicle only. The doses of hydrocortisone acetate (Merck Sharp and Dohme, Co., West Point, Pa, U.S.A.), glucagon (Eli Lilly and Co., Indianapolis, Ind., U.S.A.), thyroxine (Calbiochem, San Diego, Calif.,
684 U.S.A.) and epinephrine (Parke, Davis and Co., Detroit, Mich., U.S.A.) were 0.125, 0.003, 0.05, and 0.01 mg per fetus, respectively. Postnatal rats received intraperitoneal injections of hydrocortisone acetate or thyroxine, 2.5 and 0.003 mg per 100 g body weight, respectively. The preparations of L-phenylalanine, p-chlorophenylalanine and a-methylphenylalanine suspensions to be injected were described in a preceding paper [121. Rats were killed by decapitation. Freshly excised tissues were homogenized in four voumes of chilled 0.25 M sucrose and centrifuged at 105 000 × g for 1 h. The supernatant fluids were used for the enzyme assay. The human tissues (as described previously [4]) were processed in the same way. Phenylalanine-pyruvate aminotransferase was assayed at 30°C by the method of Spolter and Baldridge [13]. Reaction mixtures of 3.4 ml final volume contained 0.2 ml of tissue extract, sodium borate (0.58 M)/sodium arsenate (19 mM) buffer, pH 8.5, phenylalanine (58.7 raM) pyridoxal 5-phosphate (5 pg), EDTA (23.5 raM), pH 8.5, and pyruvate (44.1 mM), pH 8.5. The absorbance changes were followed in a Gilford recording spectrophotometer. Phenylalanine hydroxylase was assayed as described by McGee et al. [3] at 25°C. Enzyme activities are expressed in units (pmol per min) per g wet weight. Results The distribution of phenylalanine-pyruvate aminotransferase among some human and rat tissues is presented in Table I. The fetal human liver contained no appreciable activity; in the fetal rat the hepatic activity was 6.5 times lower than in the adult. None could be detected in the lungs {fetal or adult) of either species. The activity in rat brain was significant, about one-sixth of that in adult rat liver, and remained constant throughout development (see Fig. 1). Of the two abnormal tissues studied, rat hepatoma exhibited no phenylalaninepyruvate aminotransferase activity, and the liver of the tumor-bearing host contained about half as much as did that of normal rats of the same age. For comparison, the last two columns of Table I list some observations on phenylalanine hydroxylase. It may be seen that at the same fetal period phenylalanine hydroxylase has attained a much higher concentration (57% of that in the adult) than did phenylalanine-pyruvate aminotransferase. Another difference between the two enzymes is that only phenylalanine-pyruvate aminotransferase decreased in tumor-bearing rats; however, both enzymes were absent from the hepatoma itself. Sequential changes with age in the phenylalanine-pyruvate aminotransferase content of rat tissues are shown in Fig. 1. In kidney most of the increase occurred during postnatal life. The observations on liver differed only in detail from. those of Auerbach and Waisman [7]. We noted a multiphasic pattern. The most rapid increase occurred during the few hours before birth; this was followed by a partial loss and a second, slow phase of rise t o o k place between the 5th and 16th postnatal days. Experiments designed to identify physiological stimuli which may be responsible for the developmental formation of hepatic phenylalanine-pyruvate
685 TABLE I THE DISTRIBUTION OF PHENYLALANINE-PYRUVATE AND ADULT TISSUES OF MAN AND RAT
AMINOTRANSFERASE
AMONG
FETAL
T h e v a l u e s r e f e r t o i n d i v i d u a l s o r axe m e a n s +- S.D. o f r e s u l t s w i t h t h e n u m b e r o f t i s s u e s i n p a r e n t h e s e s . P h e n y l a l a n i n e - p y r u v a t e a m i n o t r a n s f e r a s e was m e a s u r e d in the liver a n d l u n g of 1 8 - - 2 0 w e e k h u m a n fetuses. The adult and fetal (11th--22nd week of gestation) values for human hepatic phenylalanine h y d r o x y l a s e are f r o m a p r e v i o u s p u b l i c a t i o n [ 4 ] . T h e s o l u b l e p r o t e i n c o n t e n t o f h u m a n a d u l t a n d f e t a l l i v e r w a s 1 0 2 ÷ 1 1 ( 3 ) a n d 6 8 +- 4 ( 6 ) , r e s p e c t i v e l y . T h e c o n c e p t u a l age o f r a t f e t u s e s w a s 2 0 d a y s . H o s t livers were from rats 30--45 days after the transplantation, when the weight of the hepatomas were 60-9 0 g ; t h e e n z y m e a c t i v i t i e s i n n o r m a l a d u l t l i v e r s o f t h e s e B u f f a l o r a t s w e r e t h e s a m e as t h o s e o f t h e s t r a i n u s e d i n all t h e o t h e r e x p e r i m e n t s . Tissue
Age
Enzyme activity (units/g) Phenylalanine-py ruvat e aminotransferase
Phenylalanine hydroxylase
Man
Rat
Man
0.57, 0.32 0 . 0 4 +- 0 . 0 3 (4)
1 . 3 +- 0 . 1 (9) 0 . 2 0 +- 0 . 0 2 ( 4 )
0 . 3 1 +- 0 . 0 7 ( 5 ) 0 . 1 8 -+ 0 . 0 6 ( 6 )
Liver
Adult Fetal
Lung
Adult Fetal
Brain
Adult Fetal
---
Hepatoma
Adult
--
Host liver
Adult
--
0.07
