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Comparative study on vertebrate liver AMP deaminases
Comp. Biochem. Physiol. Vol. 78B, No. 4, pp. 881-884, 1984 Printed in Great Britain
0305-0491/84 $3.00+ 0.00 © 1984PergamonPress Ltd
COMPARATIVE STUDY ON VERTEBRATE LIVER AMP DEAMINASES JOZEF SPYCHAiA Department of Biochemistry, Medical School, ul. Debinki 1, 80-211 Gdafisk, Poland (Received 10 January 1984) Abstract--1. Similar activity of AMP deaminase was found in rat, hen, turtle and flounder fiver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. 2. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. 3. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. 4. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.
INTRODUCTION Liver is the main site of urea and urate production in vertebrates. The relation of the pathway initiated by AMP deaminase (EC 3.5.4.6) to the ureotefism in the mammalian liver focused the interest of several laboratories in the last decade. Moss and MeGiven (1975) assumed that in the rat liver AMP deaminase is contributing in supplying ammonia for urea synthesis. However, Krebs et al. (1978) excluded the major role of purine nucleotide cycle in providing ammonia for urea synthesis in mammalian liver. It was also shown that adenylate catabolism in the rat liver is controlled by AMP deaminase (Van den Berghe et al., 1980), GTP being the main regulatory factor of the enzyme (Van den Berghe et al., 1977). However, the data presented recently (Spychaia et al., 1983, Spychah and Makarewicz, 1983) disclosed that a high affinity frog liver AMP deaminase is not affected by GTP and that chicken liver enzyme is not inhibited but activated by this compound. As frog, hen and rat differ considerably in the extent of ammonia, urea and urate excretion, the differences of the regulatory properties of AMP deaminase may reflect the eventual role of this enzyme in the biogenesis of the nitrogenous excretory products in these animal species. It would be tempting to relate the different regulation of AMP deamination in different animals to their ammonia-, ureo- or uricotelism. This paper presents comparison of chromatographic pattern and some regulatory properties of fiver AMP deaminase from a fish, amphibian, reptilian, bird and mammalian.
MATERIALS AND METHODS
Flounder (Platichthysflesus) was bought at a sea shore in September, frogs (Rana spp., mainly R. esculenta) were captured in September in natural environment, turtle (Testudo graeca) was a gift from Gdafisk Zoo, chickens (White leghorn) and Wistar rats were from own departmental breeding. All animals were adult and in good condition.
Isolation of fiver AMP deaminase (except flounder) was performed by the procedure described elsewhere (Stankiewicz et al., 1979) up to the stage of placing the phosphocellulose slurry with the absorbed enzyme in the column. The elution was performed using a step by step procedure with 0.625, 1.0 and 2.0 M KC1 (pH 7.0). Fractions of 3-5 ml were collected at 20 ml hr flow rate. The activity of AMP deaminase at 0.16mM AMP was assayed spectrophotometrically in 0.5cm light path length cuvettes at 265 nm in the case of frog liver extract only. Due to high phosphatase and adenosine deaminase activities in rat and chicken liver extracts, the activity of AMP deaminase there was significantly overestimated especially at low substrate concentration. At the higher substrate concentrations (0.3-10.0 raM) the enzyme activity was measured from the amount of ammonia liberated by using the phenolhypochlorite method (Chancy and Marbaeh, 1962). Microdiffusion was employed when the activity was measured in extracts (Seligson and Seligson, 1951). Incubation mixture contained 50mM cacodylate buffer, pH6.5, 100 mM KCL and 1 mM mercaptoethanol in 1.0 or 0.5 ml of total volume. Nucleotides, except ATP, were Sigma Co. products, phosphocellulose P-11 was from Whatman, imidazole and mercaptoethanol were from E. Merck AG. ATP and all the remaining reagents were supplied by Polskie Odczynniki Chemiczne POCH, Poland and were of analytical grade. RESULTS Tissue activity Table 1 presents the activity of AMP deaminase in 18,000g homogenates of the liver of flounder, frog, turtle, chicken and rat. Activities at 10.0 mM AMP for rat, chicken and flounder were similar, being an order of magnitude lower than in frog liver. The lowest activity was observed in the turtle liver extract but unfortunately there was only one individual of this species available at the time. The activity at 0.16 mM AMP concentration is presented in Table 1 only for frog fiver enzyme as at the low substrate concentration AMP deaminase activity in the rat and chicken liver could have been overestimated because of a relatively high combined activity of nonspecific phosphatases and adenosine deaminase in these tis-
881
882
JOZEF SPYCHA]'A Table 1. A M P deaminase activity in liver extracts o f several vertebrate species
Species
Activity at indicated substrate concentrations (l~moles x min i x 1g ~ of fresh tissue) 0.16 mM 10.0 mM
Rat ~5) i84_+O1~0 Chicken (6) 1.64 + 0.26 Turtle (1) 0.44 Frog (7) 3.80 + 0.32 26.8 +_3.0 Flounder (4) 1.26 + 0.31 Extracts were dialized overnight against 1.0mKCI with 10mM imidazole buffer, pH 6.8 and l mM mercaptoethanol prior to activity estimation. Data are means from the number of animals investigated given in parentheses, ±SD.
sues. The activity of the enzyme in the flounder liver extract was unmeasurable under these conditions due to a high turbidity of the sample. Nevertheless it is visible that the activity of the enzyme in the frog liver is highest among all species investigated.
Chromatographic pattern As may be seen from Fig. 1, the A M P deaminase from liver extracts of rat, chicken, turtle and flounder differ significantly in their chromatographic patterns. It has been shown previously (Spychai+a and Makarewicz, 1983) that two peaks of activity do occur in hen liver. The same is true for the flounder liver enzyme (Fig. 1). One peak of activity was observed in the extracts from rat and turtle liver as well as in the case of frog liver A M P deaminase (Spycha~a et al., 1983). Another difference is that flounder A M P deaminase did not bind to phosphocellulose in the medium containing 0.089 M phosphate buffer with 0 . 1 8 M K C 1 . It was necessary to lower the ionic strength twice to make the enzyme adsorption successful. This peculiar property was shared with liver A M P deaminase from other fish (shark and p e r c h - data not shown). Interestingly, A M P deaminase from invertebrate (crayfish tail muscle, Stankiewicz, 1982) did not bind to phosphocellulose at all.
Regulatory properties The activatory effect of 3 m M A T P on all the liver A M P deaminases studied (Table 2) was very strong at 0.3 m M A M P (except for frog liver enzyme) and seems to be substrate dependent. This effect was relatively small in the case of form I from flounder. A D P was a much weaker activator of rat and flounder A M P deaminase and did not affect the turtle liver enzyme. As shown before (Spychaia et al., 1983) this nucleotide was more effective as an activator in the case of frog liver enzyme. Most differentiated was the effect of GTP. As may be seen in Table 3 it inhibited the activity of rat and turtle enzyme, was without effect on the frog liver A M P deaminase and activated efficiently both forms of the flounder and chicken enzyme.
that a variety of enzyme forms exist in the liver of different vertebrate species. The described two forms of A M P deaminase in the liver of flounder and chicken were rather easily separable in contrast to the difficulties described by Smith et al. (1977). It cannot be excluded that also rat and other vertebrate species do possess multiple forms of this enzyme in the liver. Nevertheless at least in chicken and flounder liver the existence of the two forms can be the cause of an additional regulatory mechanism controlling the adenylate nucleotide breakdown. Van den Berghe et al. (1980) showed that G T P , inorganic phosphate and adenine nucleotides are
KcL,oM
20M+, KCL ,iRo,it Hen
I
I
t
I
r
I
4
I
I
Flounder '._;
I
I
2
8
u DISCUSSION
It has been shown recently that phosphocellulose column chromatography revealed two forms of A M P deaminase in chicken liver (Spychaia and Makarewicz, 1983). Earlier studies established that only one form of this enzyme does exist in the liver of adult rat (Smith et al., 1977). D a t a presented here indicate
60
80
Fraction N~ 120
Fig. 1. Phosphocellulose column chromatography of liver AMP deaminases.
Comparative study on vertebrate liver AMP deaminases
883
Table 2. The effect of ATP and ADP on liver AMP deaminases Substrate Reaction velocity (nmole x min-~) concentration (mM) Control 3 mM ATP 0.5 mM ADP Rat
0.3 1.0 10.0
0.15 5- 0.05 1.23 5- 0.20 15.7 5- 2.33
4.14 5- 0.77 25.05 5- 7.10 32.04 ± 7.20
Tulle 0.3 1.0 10.0
0.155-0.06 0.325-0.11 4.665-0.60
1.785-0.16 3.785-0.39 11.265-2.08
0.16±0.06 0.32±0.02 4.625-0.11
Frog 0.3 1.0 10.0
0.49 5- 0.10 2.20 5- 0.18 10.97 5- 1.90
0.56 5- 0.20 2-.17 5- 0.50 10.25 5- 1.40
0.88 5- 0.20 2.89 5- 0.60 10.83 + 1.60
Flounder I 0.3 1.0 10.0
0.22 + 0.04 0.65 5-0.10 8.02 5- 1.22
0.68 5- 0.10 1.08 5-0.22 8.50 5- 1.32
0.50 _+0.10 0.95 +0.21 8.08 __.1.82
Flounder II 0.3 1.0 10.0
0.15 5- 0.04 0.46 5- 0.09 5.62 + 0.82
3.88 5- 0.70 7.52 5- 1.20 10.20 + 1.40
0.29 5- 0.04 0.82 5- 0.10 6.20 + 1.22
The data
are m e a n s
0.59 5- 0.26 5.40 5- 1.70 15.90 5- 0.55
from three experiments, 5- SD.
Table 3. The effect of GTP on liver AMP deaminases Reaction velocity (nmoles x min-a) Enzyme source Control 0.4 mM GTP % of control Rat 0.85 + 0.11 0.26 5- 0.05 31 Chicken I 1.02 5- 0.10 2.42 5- 0.35 240 Chicken II 0.88 5- 0.12 1.22 5- 0.20 139 Turtle 1.55 + 0.25 0.12 5- 0.04 8 Frog 1.12 5- 0.10 1.05 5- 0.20 94 Flounder I 0.50 5- 0.16 0.70 5- 0.21 140 Flounder II 0.29-t-0.11 0.72+0.15 248 The activities were measured at 1.0 mM substrate concentration and the data are means from three experiments, __.SD. responsible for the regulation o f rat liver A M P d e a m i n a s e in the intact cell, G T P being assumed to be the m a i n c o m p o u n d responsible for the suppression of the enzyme activity. Recently it has been s h o w n t h a t this is n o t the case with chicken a n d frog liver A M P deaminase (Spychaia et al., 1983; Spychaia a n d Makarewicz, 1983); the former being activated a n d the latter n o t influenced by G T P . A l t h o u g h in Table 3 a c o m p a r i s o n o f the direct effect o f G T P o n enzyme activity is presented the influence o f this c o m p o u n d o n the activation exerted by A T P in different species remains to be elucidated. As m a y be seen from Table 2 A T P activates all the A M P deaminases studied a n d o n the o t h e r h a n d inorganic p h o s p h a t e inhibits at least frog, chicken a n d rat liver enzyme. G T P appears to be a regulatory factor which differentiates vertebrate liver A M P deaminases. It is assumed t h a t due to uricotelism there is m u c h higher purine nucleotide t u r n o v e r in chicken liver. Therefore it seems to be n o r e q u i r e m e n t for a such restricted regulation o f the A M P d e a m i n a t i o n as there is in the rat liver, where due to the action o f G T P a n d Pi A M P d e a m i n a s e is inhibited in 95% (Van den Berghe et al., 1977). T h e lack o f such a strong inhibition could lead to a m u c h higher activity o f the enzyme in chicken liver in vivo, especially at low
substrate concentrations. However one c a n n o t exclude the existence in chicken a n d flounder liver o f some A M P d e a m i n a s e i n h i b i t o r o t h e r t h a n G T P , which could be related r a t h e r to the overall nitrogen m e t a b o l i s m t h a n to nucleotides t u r n o v e r only. Turtle is either ureo- or uricotelic (Moyle, 1949) b u t there is some discrepancy as to which o f these p a t h w a y s predominates. It is quite possible t h a t it depends on the habitat. Therefore it seems w o r t h while to investigate the regulatory properties of A M P deaminase o f reptilian a n d o t h e r species either ureoor uricotelic in a n a t t e m p t to clarify this interesting area. One m a y assume t h a t the regulation of the liver A M P d e a m i n a t i o n can be connected with the intensity o f the overall purine nucleotide m e t a b o l i s m which in t u r n could be related to either urico- or ureotelism. Acknowledgements--The author wishes to thank Professor M. Zydowo for his interest in this work and for critical reviewing of the manuscript. This work was supported by the Ministry of Science, Higher Education and Technology within the project R.I.9., 0.1.06. REFERENCES
Chaney A. L. and Marbach E. P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130-132. Gornall A. G., Bardawill C. J. and David M. M. (1949) Determination of serum proteins by means of the buret reaction. J. biol. Chem. 177, 751-766. Krebs H. A., Hems R., Lund P., Halliday D. and Read W. W. C. (1978) Sources of ammonia for mammalian urea synthesis. Biochem. J. 176, 733-737. Moss K. M. and McGiven J. D. (1975) Characteristics of asparatate deamination by purine nucleotide cycle in the cytosol fraction of rat liver. Biochem. J. 150, 275-283. Moyle V. (1949) Nitrogenous excretion in chelonian reptiles. Biochem. J. 44, 581-585.
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JOZEF SPYCHAi'A
Seligson D. and Seligson H. (1951) A microdiffusion method for the determination of nitrogen liberated as ammonia. J. Lab. clin. Med. 38, 324-330. Smith L. D., Emerson R. L. and Knight G. D. (1977) Affinity chromatography of rat liver AMP deaminase: Evidence for single form in liver. Int. J. Biochem. 8, 883-887. Spychaia J., Stankiewicz A. and Makarewicz W. (1983) High substrate affinity and GTP insensitive AMP deaminase from frog liver. Comp. Biochem. Physiol. 74B, 851-858. Spychaia J. and Makarewicz W. (1983) Two forms of AMP deaminase in chicken liver. Biochem. Biophys. Res. Cornmun. 114, 1010-1016. Stankiewicz A., Spychaia J. Skiadanowski A. C. and Zydowo M. (1979) Comparative studies on muscle AMP
deaminase. I. Purification, molecular weight, subunit structure and metal content of the enzyme from rat, rabbit, hen, frog and pikeperch. Comp. Biochem. Physiol. 62, 363-369. Stankiewicz A. (1982) Comparative studies on AMP deaminase. VII. Purification and some properties of the enzyme from crayfish Orconectes limosus tail muscle. Comp. Biochem. Physiol. 72B, 127--132. Van den Berghe G., Bronfman M., Vanneste R. and Hers H.-G. (1977) The mechanism of adenosine triphosphate depletion in the liver after a load of fructose A kinetic study of liver adenylate deaminase. Biochem. J, 162, 601-609. Van de Berghe G., Bontemps F. and Hers H.-G. (1980) Purine catabolism in isolated rat hepatocytes. Influence of coformycine. Biochem. J. 1 ~ , 913-920.
0305-0491/84 $3.00+ 0.00 © 1984PergamonPress Ltd
COMPARATIVE STUDY ON VERTEBRATE LIVER AMP DEAMINASES JOZEF SPYCHAiA Department of Biochemistry, Medical School, ul. Debinki 1, 80-211 Gdafisk, Poland (Received 10 January 1984) Abstract--1. Similar activity of AMP deaminase was found in rat, hen, turtle and flounder fiver when estimated at high AMP concentration. The enzyme activity was of an order of magnitude higher in frog liver. 2. Simple step by step phosphocellulose column chromatography revealed two forms of AMP deaminase in chicken and flounder liver and one form in the liver of rat and turtle. 3. All enzymes (except for frog liver AMP deaminase) were activated by ATP. The enzymes from rat, frog and both forms from flounder were also activated by ADP. 4. GTP exhibited a variety of effects. The enzyme from rat and turtle was inhibited, both forms from hen and flounder were activated and frog liver enzyme was not influenced.
INTRODUCTION Liver is the main site of urea and urate production in vertebrates. The relation of the pathway initiated by AMP deaminase (EC 3.5.4.6) to the ureotefism in the mammalian liver focused the interest of several laboratories in the last decade. Moss and MeGiven (1975) assumed that in the rat liver AMP deaminase is contributing in supplying ammonia for urea synthesis. However, Krebs et al. (1978) excluded the major role of purine nucleotide cycle in providing ammonia for urea synthesis in mammalian liver. It was also shown that adenylate catabolism in the rat liver is controlled by AMP deaminase (Van den Berghe et al., 1980), GTP being the main regulatory factor of the enzyme (Van den Berghe et al., 1977). However, the data presented recently (Spychaia et al., 1983, Spychah and Makarewicz, 1983) disclosed that a high affinity frog liver AMP deaminase is not affected by GTP and that chicken liver enzyme is not inhibited but activated by this compound. As frog, hen and rat differ considerably in the extent of ammonia, urea and urate excretion, the differences of the regulatory properties of AMP deaminase may reflect the eventual role of this enzyme in the biogenesis of the nitrogenous excretory products in these animal species. It would be tempting to relate the different regulation of AMP deamination in different animals to their ammonia-, ureo- or uricotelism. This paper presents comparison of chromatographic pattern and some regulatory properties of fiver AMP deaminase from a fish, amphibian, reptilian, bird and mammalian.
MATERIALS AND METHODS
Flounder (Platichthysflesus) was bought at a sea shore in September, frogs (Rana spp., mainly R. esculenta) were captured in September in natural environment, turtle (Testudo graeca) was a gift from Gdafisk Zoo, chickens (White leghorn) and Wistar rats were from own departmental breeding. All animals were adult and in good condition.
Isolation of fiver AMP deaminase (except flounder) was performed by the procedure described elsewhere (Stankiewicz et al., 1979) up to the stage of placing the phosphocellulose slurry with the absorbed enzyme in the column. The elution was performed using a step by step procedure with 0.625, 1.0 and 2.0 M KC1 (pH 7.0). Fractions of 3-5 ml were collected at 20 ml hr flow rate. The activity of AMP deaminase at 0.16mM AMP was assayed spectrophotometrically in 0.5cm light path length cuvettes at 265 nm in the case of frog liver extract only. Due to high phosphatase and adenosine deaminase activities in rat and chicken liver extracts, the activity of AMP deaminase there was significantly overestimated especially at low substrate concentration. At the higher substrate concentrations (0.3-10.0 raM) the enzyme activity was measured from the amount of ammonia liberated by using the phenolhypochlorite method (Chancy and Marbaeh, 1962). Microdiffusion was employed when the activity was measured in extracts (Seligson and Seligson, 1951). Incubation mixture contained 50mM cacodylate buffer, pH6.5, 100 mM KCL and 1 mM mercaptoethanol in 1.0 or 0.5 ml of total volume. Nucleotides, except ATP, were Sigma Co. products, phosphocellulose P-11 was from Whatman, imidazole and mercaptoethanol were from E. Merck AG. ATP and all the remaining reagents were supplied by Polskie Odczynniki Chemiczne POCH, Poland and were of analytical grade. RESULTS Tissue activity Table 1 presents the activity of AMP deaminase in 18,000g homogenates of the liver of flounder, frog, turtle, chicken and rat. Activities at 10.0 mM AMP for rat, chicken and flounder were similar, being an order of magnitude lower than in frog liver. The lowest activity was observed in the turtle liver extract but unfortunately there was only one individual of this species available at the time. The activity at 0.16 mM AMP concentration is presented in Table 1 only for frog fiver enzyme as at the low substrate concentration AMP deaminase activity in the rat and chicken liver could have been overestimated because of a relatively high combined activity of nonspecific phosphatases and adenosine deaminase in these tis-
881
882
JOZEF SPYCHA]'A Table 1. A M P deaminase activity in liver extracts o f several vertebrate species
Species
Activity at indicated substrate concentrations (l~moles x min i x 1g ~ of fresh tissue) 0.16 mM 10.0 mM
Rat ~5) i84_+O1~0 Chicken (6) 1.64 + 0.26 Turtle (1) 0.44 Frog (7) 3.80 + 0.32 26.8 +_3.0 Flounder (4) 1.26 + 0.31 Extracts were dialized overnight against 1.0mKCI with 10mM imidazole buffer, pH 6.8 and l mM mercaptoethanol prior to activity estimation. Data are means from the number of animals investigated given in parentheses, ±SD.
sues. The activity of the enzyme in the flounder liver extract was unmeasurable under these conditions due to a high turbidity of the sample. Nevertheless it is visible that the activity of the enzyme in the frog liver is highest among all species investigated.
Chromatographic pattern As may be seen from Fig. 1, the A M P deaminase from liver extracts of rat, chicken, turtle and flounder differ significantly in their chromatographic patterns. It has been shown previously (Spychai+a and Makarewicz, 1983) that two peaks of activity do occur in hen liver. The same is true for the flounder liver enzyme (Fig. 1). One peak of activity was observed in the extracts from rat and turtle liver as well as in the case of frog liver A M P deaminase (Spycha~a et al., 1983). Another difference is that flounder A M P deaminase did not bind to phosphocellulose in the medium containing 0.089 M phosphate buffer with 0 . 1 8 M K C 1 . It was necessary to lower the ionic strength twice to make the enzyme adsorption successful. This peculiar property was shared with liver A M P deaminase from other fish (shark and p e r c h - data not shown). Interestingly, A M P deaminase from invertebrate (crayfish tail muscle, Stankiewicz, 1982) did not bind to phosphocellulose at all.
Regulatory properties The activatory effect of 3 m M A T P on all the liver A M P deaminases studied (Table 2) was very strong at 0.3 m M A M P (except for frog liver enzyme) and seems to be substrate dependent. This effect was relatively small in the case of form I from flounder. A D P was a much weaker activator of rat and flounder A M P deaminase and did not affect the turtle liver enzyme. As shown before (Spychaia et al., 1983) this nucleotide was more effective as an activator in the case of frog liver enzyme. Most differentiated was the effect of GTP. As may be seen in Table 3 it inhibited the activity of rat and turtle enzyme, was without effect on the frog liver A M P deaminase and activated efficiently both forms of the flounder and chicken enzyme.
that a variety of enzyme forms exist in the liver of different vertebrate species. The described two forms of A M P deaminase in the liver of flounder and chicken were rather easily separable in contrast to the difficulties described by Smith et al. (1977). It cannot be excluded that also rat and other vertebrate species do possess multiple forms of this enzyme in the liver. Nevertheless at least in chicken and flounder liver the existence of the two forms can be the cause of an additional regulatory mechanism controlling the adenylate nucleotide breakdown. Van den Berghe et al. (1980) showed that G T P , inorganic phosphate and adenine nucleotides are
KcL,oM
20M+, KCL ,iRo,it Hen
I
I
t
I
r
I
4
I
I
Flounder '._;
I
I
2
8
u DISCUSSION
It has been shown recently that phosphocellulose column chromatography revealed two forms of A M P deaminase in chicken liver (Spychaia and Makarewicz, 1983). Earlier studies established that only one form of this enzyme does exist in the liver of adult rat (Smith et al., 1977). D a t a presented here indicate
60
80
Fraction N~ 120
Fig. 1. Phosphocellulose column chromatography of liver AMP deaminases.
Comparative study on vertebrate liver AMP deaminases
883
Table 2. The effect of ATP and ADP on liver AMP deaminases Substrate Reaction velocity (nmole x min-~) concentration (mM) Control 3 mM ATP 0.5 mM ADP Rat
0.3 1.0 10.0
0.15 5- 0.05 1.23 5- 0.20 15.7 5- 2.33
4.14 5- 0.77 25.05 5- 7.10 32.04 ± 7.20
Tulle 0.3 1.0 10.0
0.155-0.06 0.325-0.11 4.665-0.60
1.785-0.16 3.785-0.39 11.265-2.08
0.16±0.06 0.32±0.02 4.625-0.11
Frog 0.3 1.0 10.0
0.49 5- 0.10 2.20 5- 0.18 10.97 5- 1.90
0.56 5- 0.20 2-.17 5- 0.50 10.25 5- 1.40
0.88 5- 0.20 2.89 5- 0.60 10.83 + 1.60
Flounder I 0.3 1.0 10.0
0.22 + 0.04 0.65 5-0.10 8.02 5- 1.22
0.68 5- 0.10 1.08 5-0.22 8.50 5- 1.32
0.50 _+0.10 0.95 +0.21 8.08 __.1.82
Flounder II 0.3 1.0 10.0
0.15 5- 0.04 0.46 5- 0.09 5.62 + 0.82
3.88 5- 0.70 7.52 5- 1.20 10.20 + 1.40
0.29 5- 0.04 0.82 5- 0.10 6.20 + 1.22
The data
are m e a n s
0.59 5- 0.26 5.40 5- 1.70 15.90 5- 0.55
from three experiments, 5- SD.
Table 3. The effect of GTP on liver AMP deaminases Reaction velocity (nmoles x min-a) Enzyme source Control 0.4 mM GTP % of control Rat 0.85 + 0.11 0.26 5- 0.05 31 Chicken I 1.02 5- 0.10 2.42 5- 0.35 240 Chicken II 0.88 5- 0.12 1.22 5- 0.20 139 Turtle 1.55 + 0.25 0.12 5- 0.04 8 Frog 1.12 5- 0.10 1.05 5- 0.20 94 Flounder I 0.50 5- 0.16 0.70 5- 0.21 140 Flounder II 0.29-t-0.11 0.72+0.15 248 The activities were measured at 1.0 mM substrate concentration and the data are means from three experiments, __.SD. responsible for the regulation o f rat liver A M P d e a m i n a s e in the intact cell, G T P being assumed to be the m a i n c o m p o u n d responsible for the suppression of the enzyme activity. Recently it has been s h o w n t h a t this is n o t the case with chicken a n d frog liver A M P deaminase (Spychaia et al., 1983; Spychaia a n d Makarewicz, 1983); the former being activated a n d the latter n o t influenced by G T P . A l t h o u g h in Table 3 a c o m p a r i s o n o f the direct effect o f G T P o n enzyme activity is presented the influence o f this c o m p o u n d o n the activation exerted by A T P in different species remains to be elucidated. As m a y be seen from Table 2 A T P activates all the A M P deaminases studied a n d o n the o t h e r h a n d inorganic p h o s p h a t e inhibits at least frog, chicken a n d rat liver enzyme. G T P appears to be a regulatory factor which differentiates vertebrate liver A M P deaminases. It is assumed t h a t due to uricotelism there is m u c h higher purine nucleotide t u r n o v e r in chicken liver. Therefore it seems to be n o r e q u i r e m e n t for a such restricted regulation o f the A M P d e a m i n a t i o n as there is in the rat liver, where due to the action o f G T P a n d Pi A M P d e a m i n a s e is inhibited in 95% (Van den Berghe et al., 1977). T h e lack o f such a strong inhibition could lead to a m u c h higher activity o f the enzyme in chicken liver in vivo, especially at low
substrate concentrations. However one c a n n o t exclude the existence in chicken a n d flounder liver o f some A M P d e a m i n a s e i n h i b i t o r o t h e r t h a n G T P , which could be related r a t h e r to the overall nitrogen m e t a b o l i s m t h a n to nucleotides t u r n o v e r only. Turtle is either ureo- or uricotelic (Moyle, 1949) b u t there is some discrepancy as to which o f these p a t h w a y s predominates. It is quite possible t h a t it depends on the habitat. Therefore it seems w o r t h while to investigate the regulatory properties of A M P deaminase o f reptilian a n d o t h e r species either ureoor uricotelic in a n a t t e m p t to clarify this interesting area. One m a y assume t h a t the regulation of the liver A M P d e a m i n a t i o n can be connected with the intensity o f the overall purine nucleotide m e t a b o l i s m which in t u r n could be related to either urico- or ureotelism. Acknowledgements--The author wishes to thank Professor M. Zydowo for his interest in this work and for critical reviewing of the manuscript. This work was supported by the Ministry of Science, Higher Education and Technology within the project R.I.9., 0.1.06. REFERENCES
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