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Intestinal arginase in vertebrates and invertebrates
Corap. Biochem. Physiol., Vol. 6lB, pp. 545 to 552 © Pergamon Press Ltd 1978. Printed in Great Britain
0305-0491/78/1115-0545502.00/0
INTESTINAL ARGINASE IN VERTEBRATES AND INVERTEBRATES B. LISOWSKA-MYJAK,L. TOMASZEWSKi a n d L. HRYCKIEWICZ Medical School of Warsaw, Department of Clinical Biochemistry, Institute of Biopharmacy, Regional Veterinary Station Koszalin, 02-097 Warszawa, ul. Banacha 1, Poland
(Received 24 October 1977) Abstract--l. Arginase was found to be present in the intestine in all species of Annelida, Arthropoda and Chordata studied. 2. The activity of intestinal arginase differs from species to species, the differences reaching two orders of magnitude (100 × ). 3. The highest activity of intestinal arginase was observed in the rodents (mouse, rat, hamster). 4. In animals in which the enzyme activity was high or moderately high, arginase activity showed topographical differentiation along the long axis of the intestine.
As a rule, samples of the mucosa for the experiments were taken from the proximal, middle and distal segments of the intestine; the only exceptions were the earthworm, crayfish, cockroach, locust and frog.
INTRODUCTION There are but scant data on the occurrence of arginase activity in intestinal mucosa. So far, the presence of this enzyme was d e m o n s t r a t e d in intestinal m u c o s a of the e a r t h w o r m (Lumbricus terrestris) (Reddy & Campbell 1968), dog (Kossell & Dakin, 1904; K o n a r s k a & Tomaszewski, 1975b), mouse (Greenstein & T h o m p s o n , 1943; Kochakian, 1945), rat (Greengard et al., 1970; K o n a r s k a & Tomaszewski, 1975a), ox (Kossel & Dakin, 1904) and man ( K o n a r s k a & Tomaszewski, 1975b). Arginase was reported to be present in the stomach of the fish Raia clavata (Baret et al., 1966) but its intestine has not been checked for the enzyme activity. This paper presents the results of research on the activity of intestinal arginase in representatives of vertebrates a n d invertebrates.
Homogenates Suspensions of the mucosa or the whole intestine were homogenized in a glass Potter Elvehjem homogenizer, for 1.5 rain at 1900 rev/min. Determination of arginase activity The enzyme activity was determined by measuring the increase in ornithine formed. The homogenates were diluted with 5 m M Tris-HCl buffer, pH 7.5 (to which manganese was not added). The incubation mixture contained (per ml) 20pmoles of arginine, I/tmole of MnCI2, 50/~moles of Tris-HCl buffer pH 9.5, the enzyme preparation, and deionized water to bring the volume to 1 ml. The samples contained 8-304/~g of protein ; in the majority of samples this value ranged from 50 to 150pg. The samples were incubated at 37°C for 15 rain. The reaction was stopped by placing the tubes in a boiling-water bath for 10 rain. The ornithine formed was determined according to Chinard (1952).
MATERIALS AND METHODS
Animals In most animals studied the intestine, isolated immediately after killing of the animal, was cut lengthwise, the intestinal contents were removed and the tissue was linsed and frozen. After 2 7 days, the mucosa was scraped off with a scalpel, weighed and suspended in 4 vol of 1 mM MnCI2 5 mM Tris-HCl buffer. This procedure was applied to herring, salmon, bream, cod, pike-perch, duck, hen, rabbit, coypu, guinea-pig, mink, fox, horse, hog, hippopotamus, ox, sheep, antelope, as well as to human intestine obtained from the Department of Pathological Anatomy. The intestines of the frog, pigeon, mouse, rat and hamster was not subjected to freezing, and the mucosa was suspended in the buffer as soon as possible after killing of the animals. These animals were killed by decapitation. Since it proved impossible to isolate mucosa of small animals (earthworm, crayfish, cockroach, locust) the whole intestine wall was disintegrated, suspended in the buffer and pooled samples were examined. The earthworm's intestine was homogenized immediately after being suspended in the buffer; the intestines of the crayfish, cockroach and locust were frozen; they were homogenized not later than 2 days after.
Protein determination Protein was assyed by the method of Lowry et al. (1951 t, bovine serum albumin being used as a standard. RESULTS The activity of intestinal arginase was determined in 30 animal species, including five invertebrate and 25 vertebrate species. A m o n g the latter, the majority were m a m m a l s (15 species). The results presented in Table 1 are arranged in the order of phylogenetic development of the animals studied. The activities of intestinal arginase in columns 4 a n d 5 are expressed, respectively, as ~moles of o r n i t h i n e / m g of protein per 15 rain and #moles of ornithine/g of mucosa per min. 1. Analysis of the presented data indicates that in the animals studied the enzyme activity shows large variations (by a b o u t two orders of magnitude). In the studied invertebrate species (crayfish, cockroach, locust) the activity is very low, ranging from 0.11 to
545
B. LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
546
Table 1. The activities of intestinal arginase (mean and range) in studied species Number of samples 3
No. I 1
2
3 4 5
6 7 8 9 10
II
12 12a 13 14
15
16 17 18 19 20 21 22 23 24 25
Type: Annelida Lumbricus terrestris (earthworm) Type: Arthropoda Class: Crustacea Astatus (crayfish) Class: Insecta Periplaneta orientalis (cockroach) Acridium migratorius (locust) Type: Chordata Class: Pisces Clupea harenyus (herring) Sahno salar (salmon) Ahramis brama (bream) Gadus callarias (cod) Lucioperca (pike perch) Class: Amphibia Rana esculenta (frog) Class: Ayes Anser domesticus (duck) Callus domesticus (hen) B toiler Columbia lieia (Pigeon) Class : Mammalia Order: Lagomorpha Oryctalogus (rabbit) Order : Rodentia M yopotamus coypus (coypu) Cavia parcellus (guinea-pig) Mus (mouse) Rattus rattus (rat) Cricetus vulyaris (hamster) Order: Carnivora Lutreola lutreola (mink) Canis vulpes (fox) Canis (dog) Order : Perissodactyla Equus (horse) Order: Artiodactyla Sus domestica (hog) Hippopotamus amphibius (hippopotamus)
4
0.19 1.56
1. t9-16.5
4
0.40 0.36-0.45
1.45 1.21 1.73
3
0.16 0.14-0.18 0.11 0.084). 15
1.03 0.97-1.11 0.92 0.66-1.37
1
0.26 0.24-0.29 0.27
1.08 0.84-1.45 1.06
1
0.24
0.92
3
0.59 0.524).63 0.44 0.42-0.48
1.89 1.38-2.29 1.81 1.69-1.92
3
0.17 0.144).20
0.77 0.70-0.83
5
0.10 0.064). 15 0.13 0.09-0.17 0.12 0.114).15 0.69 0.37-1.04
0.80 0.39-1.58 0.72 0.53-0.82 1.41 1.12-1.85 3.53 1.53-4.73
2.23 1.84-2.69
14.41 12.00~ 18.40
1.15 0.89-1.42 1.07 0.91-1.36 9.26 5.48 12.13 5.49 3.72-8.40 7.61 6.12-9.89
4.69 3.10-5.73 5.10 3.22-8.41 100.43 76.45-139.00 43.99 36.22-53.31 84.47 63.80--129.58
0.38 0.22-0.50 0.29 0.21-0.35 0.34 0.12-0.51
1.36 0.88-1.66 1.65 1.35-2.52 3.30 1.00-4.90
0.07 0.06-0.07
0.40 0.38-0.42
3
3
3
3 3 4
3 4 5 6 3 4 5 8 5
3
7 1
Activity pM ornithine/mg /~M ornithine/g prot. per 15 min tissue per 1 min 4 5
0.63 0.53-0.76 0.06
4.51 3.94-5.25 0.23
(continued)
Intestinal arginase in vertebrates and invertebrates
547
Table 1. contd.
No. 1
2
26
Bos taurus
Number of samples 3 6
(oxt 27
Ovis
2
(sheep) 28 29
!
Antelope
(antelope) Order: Primates Homo sapiens
20
(many)
0.40/~moles of ornithine/mg of protein per 15 min. In the lowest vertebrates studied (salmon, bream, cod, pike-perch, herring) the activity is also very low, practically the same as in amphibians (frog) and birds (duck, hen, pigeon). In this group, rather low activity was observed only in the cod and pike-perch (0.44-0.59#moles of ornithine/mg of protein per 15 min). In the frog, hen and duck the activity was very low (0.10-0.17 pmoles of ornithine/mg of protein per 15min), whereas in the pigeon the activity was almost five times as high. Increased arginase activity was observed only in some of the mammals, namely the rabbit, coypu and guinea-pig. It was by a factor of 5-10 higher than the activity in animals at the lower grades of phylogenetic development. On the other hand, distinctly higher arginase activity was observed in some of the rodents, i.e. in the mouse, rat and hamster. The average enzyme activity in Carnivora (fox, mink) is distinctly lower than in rodents. In the species of higher animals the average enzyme activity is low, not exceeding 0.17 pmoles of ornithine/mg of protein per 15 min, except in the Artiodactylous hog and in man, which show moderate activity. 2. Comparison of the activity expressed in pmoles of ornithine/mg of protein per 15min with that expressed as #moles of ornithine/g of mucosa per 1 min, shows that the patterns obtained are very similar. The alternative form of expressing the results does not provide new information as compared with the activity calculated per 1 mg of protein. Our results are at variance with those presented by Dixon & Webb (1964). We found rather high activity of intestinal arginase in the rabbit, guinea-pig and rat, whereas in mouse intestine the observed enzyme activity was not as high as that reported by Dixon & Webb (1964). 3. The results discussed above were average values of arginase activity in the intestines of the animals studied. However, we have also observed differences dependent on the intestinal segment in which the activity was determined. The activity of arginase presented in Table 2 for each animal relates to three segments of the intestine: the proximal, middle and distal. In lower species, in which the average activity is very low or rather low, the activity is the same over the whole length of the intestine (herring, salmon, bream, pike-perch, duck, hen). Beginning with the pigeon, there is a distinct differentiation of
Activity #M ornithine/mg pM ornithine/g prot. per 15 sec tissue per 1 see 4 5 0.12 0.08q).23 0.17 0.12~3.21 0.09
0.86 0.44- 2.14 1.18 0.7(~1.65 0.44
0.38 0.244).52
1.09 0.56 1.62
arginase activity with the topographical location of the segment studied. As a rule, the activity is highest in the segment proximal to the stomach (duodenum), than it decreases, the steepness of the gradient differing from species to species (Fig. 1). This phenomenon is observed in all the species studied up to the level of the hippopotamus, and is observed only in those species in which the arginase activity is high or moderate. In herbivorous animals this topographical differentiation was not observed. 4. The results obtained for the earthworm and the snail call for some additional comments. We have performed at least 10 separate measurements of the intestinal arginase activity in the earthworm, under a variety of conditions: immediately after collecting the animals and on animals kept for differing time periods at the laboratory. The results were highly divergent: from 0.19 to 1.56 #moles of ornithine/mg of protein per 15 min. The earthworms studied were not kept under the same conditions: some of them were kept for a few days in the laboratory, and a part were killed within a short time after digging them out from deeper layers of the soil. These conditions had a distinct effect on the arginase activity: the earthworms kept in the laboratory (fasted) showed an activity of 1.14-1.56/~moles of ornithine/mg of protein per 15 min, which corresponds to 764-990#moles of ornithine/g of mucosa per hr, whereas in the freshly collected specimens the activity ranged from 0.043 to 0.42 #moles of ornithine/mg of protein per 15 min, which corresponds to 18.6-172/~moles of ornithine/g of mucosa per hr. Our observations are in agreement with the similarly expressed data of Campbell (1970a). The activity of intestinal arginase in the snail Helix pomatia is not included in table due to the difficulty in separating the intestine from the liver in this organism. No differences in intestinal arginase activity were observed between the common household hen and the broiler chicken.
DISCUSSION
The activity of intestinal arginase was studied in 30 animal species belonging to the types Annelida, Arthropoda and Chordata. Analysis of the data presented in Table 1 shows large variations of the enzyme
B. LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
548
Table 2. The topographical differentiation of arginase activity along the long axis of the intestine in studied species. Parts of intestine: I, postventricular part; II, middle part; III, distal part; IV, colon Arginase activity
No.
Species
1 Type: Annelida Lumbricus terrestris 2 Type: Arthropoda Class: Crustacea Astacus 3 Class: Insecta Periplaneta orientalis 4 Acridium migratorius 5 Type: Chordata Class : Pisces Clupea harengus 6 Salmo salar 7 Abramis brama 8 Gadus callarias 9 Lucioperca 10 Class: Amphibia Rana esculenta 11 Class : Ayes A riser domesticus 12 Gallus domesticus 12 Broiler 13 Columbia livia 14 Class: Mammalia Order: Lagomorpha Oryctaloous 15 Order: Rodentia Myopotamus coypus 16 Caz;ia parcellus 17 Mus 18 Rattus rattus 19 Cricetus vulgaris 20 Order : Carnivora Lutreola lutreola 21 Canis vulpes 22 Canis 23 Order: Perissodactyla Equus 24 Order : Artiodactyla Sus domestica 25 Hippopotamus amphibius 26 Bos taurus 27 Ovis 28 Antelope 29 Order : Primates Homo sapiens
~M ornithine/mg prot. per 15 min Parts of intestine I I1 tll IV
~M ornithine/g tissue per 1 min Parts of intestine i II III IV
Whole intestine 0.19 1.56
Whole intestine 1.19-16.50
Whole intestine 0.40
Whole intestine 1.45
Whole intestine 0.16 Whole intestine 0.11
Whole intestine 1.03 Whole intestine 0.92
0.27 0.24 0.28 0.59 0.42
0.25 0.21 0.21 0.52 0.46
1.25 0.64 1.26 2.56 1.83
0.26 0.36 -0.66 -
0.97 1.18 0.57 1.84 1.78
0.81 1.36 -1.47
Whole intestine 0.77
Whole intestine 0.17
0.94 0.84 1.38 5.16
0.78 0.75 1.20 3.60
0.48 0.57 0.99 0.63
17.25
14.18
12.39
9.95
6.85 10.68 137.73 55.70 118.80
2.35 6.96 139.89 68.45 98.35
0.51 86.53 22.48 75.30
-37.55 10.70 9.27
4.90
2.01 3.68 4.00
1.44 1.05 1.00
0.64 0.09
0.03
0.67
0.57
0.27
0.16
0.49
0.12
-
9.63
2.88
0.67
0. 30 0.09 0.13 O. I 1
0.04 0.07 0.12 0.05
0.38
0.10 0.11 O.12
0.13 0.54 0.92 0.61
0.60 0.67 0.48
0.19 0.30 0.50 0.23
0.24
0.20
0.94
0.54
O.13 O. 15 0.13 1.14
0.10 O. 12 0.10 0.77
0.09 O. 12 0.09 0.21
2.62
2.18
1.93
1.58 1.94 9.39 6.13 8.16
0.72 1.51 17.67 7.23 14.21
0.12 7.33 2.02 6.26 0.41 0.32 0.12
0.23 0.03
0.51
0.50 0.55 0.40
O.10
0.09
0.06
1.40 0.10 -~ 0.70
activity in these types of animals; four groups may be discerned: 1. Animals with very low arginase activity, up to 0.20/~moles of ornithine/mg of protein per 15 min; this group comprises both phylogenetically lower and higher animals: cockroach, locust, frog, duck, domestic hen, broiler chicken, horse, hippopotamus, ox, sheep and antelope. 2. Animals with rather low activity: 0.220.60pmoles of ornithine/mg of protein per 15min: crayfish, herring, salmon, bream, cod, pike-perch, mink, fox, dog and man. 3. Animals with moderate activity; 0.63-1.15pmoles of ornithine/mg of protein per 15rain: pigeon, coypu, guinea-pig, hog.
k~
1.90 0.99 0.77 1.79
1.80
4. Animals with high activity: 1.15-9.26/~moles of ornithine/mg of protein per 15 min : rabbit, rat, hamster, mouse. We are well aware of arbitrary criteria of this division. However, large differences in arginase activity (even by two orders of magnitude) seemed to justify our choice. The group with the highest activity is most distinctly separated from the others, which is clearly evident from Fig. 2. There is a smooth transition between average values of the enzyme activity for the particular species, a small gradient of the decrease or increase in activity being observed except for the rabbit, rat, hamster and mouse (Fig. 2). The very low or rather low activity is found both in lower and higher animals. Thus, it seems possible to sup-
Intestinal arginase in vertebrates and invertebrates
549
18
14 13 12 II
I0
._c E
9 8
tO
A
7 i:x
\ BA
6
o
% ¢:
;:L
2.6 25 24 2:3 22 21 20 19 1.8 1.7 1,6 1,5 1,4 13 1,2 I.I 1.0 0.9 08 0.7 0.6 05 0,4 0:3 0.2 O I 0
AA
A
A
IA
Ax A +A
I
K
TIT
IK
Fig. 1. The topographical differentiation of arginase activity in different parts of intestine: I, postventricular part; II, middle part; III, distal part; IV, colon. Studied species: mouse OA, hamster OA, rat /XA, rabbit &A, guinea-pig DA, coypu IA, hog xA, pigeon +A, mink O, fox 0, man A, dog &, cod D, pike-perch x, salmon +, bream OB, herring liB, duck, hen, hippopotamus, ox, sheep, antelope 1.
pose that higher animals had a higher activity of intestinal arginase, which subsequently they had lost or that the rodents selectively elaborated, for unknown reasons, high activities. Moreover, it should be noted that the activity of intestinal arginase showed topographical differences. The results presented permit us to discern in this respect two groups of animals: (1)--those in which arginase activity is the same over the whole length of the intestine, irrespective of the level of the enzyme activity (herring, salmon, bream, cod, pike-perch, duck, domestic hen, broiler chicken, horse, hippopotamus, ox, sheep, antelope); (II)----those in which there is a distinct topographical differentiation of the activity (Fig. 1) which is the
highest in jejunum and decreases gradually, with differing gradients, toward the distal end of the intestine (pigeon, rabbit, coypu, guinea-pig, mouse, rat, hamster, dog, fox, mink, hog, man). It should be noted that in the present study, only adult animals were used. Arginase activity is also known to depend on the stage of ontogenetic development (rat, man, Fasciola hepatica). The activity of intestinal arginase in the rat up to the second week of life is low (of the order of 1-2/~moles of ornithine/mg of protein per 15 min). An increase in the activity is observed between the second and fourth week, adult values being reached at 5 weeks of life (Konarska & Tomaszewski, 1975a). Similarly, it was found that the activity of intestinal arginase is lower
550
B, LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
13
12
10
90
8.0 .E E
70
o.
60 Q. 50
o
40
30 >7 >
n-
28 26 24 22 20 18 16 14 12 ~ o
I0 09 08 0.7 06 0.5 04 0.3 02 OI
1
-
-~.c
-
o
-
ocn
o
u,~
o ~ ¢: o
..~
"~EE
iE
00~
m
°
o~
i1)
, , , , o, o4 1
*~
oo
~o
,
Fig. 2. The comparison of the means of activities of intestinal arginase in species arranged in phylogenetic development.
in children in the first year of life than in adults (Konarska & Tomaszewski, 1975a). On the other hand, in Fasciola hepatica no arginase activity was found in the eggs, miracidium and metacercaria, but was found in the youthful, 15-day-old form of the fluke (Campbell, 1970a). The studies performed so far on the urea cycle limited mainly to two organs: liver and kidney (Hirsh-Kolb & Greenberg, 1968; Porembska et al.,
1971). The functioning of the full urea cycle is most efficient in liver. The discovery of inborn errors of metabolism and some other clinical observations pointed to the possibility of extrahepatal urea formation (Konarska & Tomaszewski, 1975a). Florkin & Stotz (1975a) considers that ureogenesis in various animal species plays a role in protection against changes in osmotic pressure of the environment. Thus, it would follow that the efficiency of the
Intestinal arginase in vertebrates and invertebrates urea cycle in salt-watcr fish should be greater than in freshwater ones. In our experiments both salt-water species (cod, herring) and species living in brackish water (salmon, bream, pike-perch) were studied but the results obtained showed no distinct differences between these groups, and thus did not confirm Florkin & Stotz's view, at least as concerns intestinal arginase. At this stage of our work we have no data on the enzyme activity in the intestine of freshwater fish, and we are unable to characterize the intestinal arginase with respect to different osmotic conditions. In analysing the activity of intestinal arginase in various animal species one should not disregard the effect of feeding. The fasted earthworm is known to have higher enzyme activity than the fed animal (Campbell, 1970b). Our experiments included another example of wide differences in feeding of the same species: the domestic hen and artificially fed broiler chick. These differences, however, did not affect either the level of the activity or its topographical distribution. Thus it seems that in the hen activity of intestinal arginase follows a species-dependent pattern. Topographical differentiation of the intestinal arginase activity in animals with moderate and high enzyme activity (Fig. 1) is of considerable interest. It is not clear whether this differentiation is a speciesdependent property, or is a secondary trait formed due to differences in the type of feeding. However, there are no data which would permit elucidation of this question. The observed differences in the intestinal arginase activity in various animal species are not an isolated phenomenon; also the hepatic arginase is known to show species-dependent differences (Campbell, 1970c), e.g. the activity expressed as units/gram of wet tissue weight was 1000 in the frog, 658 in lion, 458 in ox and 388 in rat. Similar differences in the activity of hepatic arginase were found in birds, e.g. 220 units in the kingfisher, but no activity at all in pigeon. The presented data permit to state that the differences in the arginase activity in various animals are speciesdependent, genetically determined traits. It seems rather striking that no arginase activity was found in pigeon liver, whereas in pigeon intestine the activity reached moderate values. In considering the results obtained one should take into account differences in the functioning of arginase at the particular level of phylogenetic development. Arginase lowers the efficiency of arginine biosynthesis which supplies the body with this exogenous amino acid; it leads also to formation of other metabolites (glutamic acid, proline, polyamines) and of urea indispensable for osmotic regulation. Our work contains several new observations, which lead to a number of unresolved questions. First, is high activity of intestinal arginase an isolated phenomenon, or is it accompanied by an increase in the activity of other enzymes of the urea cycle and increase in its overall efficiency? As concerns higher animals, there are no experimental data permitting us to answer this question. If it is an isolated phenomenon, independent of the activity of other enzymes of the urea cycle, one could expect that intestinal arginase plays a special role in the body of these animals. It seems impossible to reject the assumption
551
that the role of intestinal arginase is not limited to detoxication of ammonia, but would also include formation of urea by by-passing ammonia binding. We are unable to explain why intestinal arginase activity is high in rodents and the rabbit (La#omorpha). Is their metabolism so widely different that it requires additional formation of urea in the intestinal mucosa? F r o m observations of pathological cases we know that in man the increase in the activity of intestinal arginase may lead to an increase in the extracellular pool of urea (Konarska & Tomaszewski, 1975a). The aim of our work was a very modest and limited one: to inquire into the activity of arginase in phylogenetic development, in a tissue not typical for ureogenesis, i.e. the intestine. We have demonstrated very wide differences in the enzyme activity and its topographical differentiation. These facts point to a number of unresolved problems, which may shed new light on the alternative role of arginase as a separate source of urea formation, a function not related to the role of urea cycle in the metabolism of ammonia.
REFERENCES BARET R., MOURGUE• PELLEGRINJ. (1966) Sur l'existence de deux activit6s arginasiques dans les tissus hepatique, r6nal et stomacal de la raie clout6e. C. R. Sdanc. Soe. biol. 160, 1796-1800. CAMPBELL J. W. (1970a) Comparative Biochemistry oJ Nitrogen Metabolism. Vol. 1, The Invertebrates, p. 84, Academic Press, New York. CAMPBELL J. W. (1970b) Comparative Biochemistry oJ Nitrogen Metabolism. Vol 1, The Invertebrates, p. 256. Academic Press, New York. CAMPBELL J. W. (1970c) Comparative Biochemistry oJ Nitrooen Metabolism. Vol. 2, The vertebrates, p. 750. Academic Press, New York. CmNARD F. P. (1952) Photometric estimation of proline and ornithine J. biol. Chem. 199, 91-95. DIXON M. & WEBB E. (1964) Enzymes pp. 636-645. Longmans, Green, London. FLORKIN M. & STOTZ E. H. (1975a) Comprehensive Biochemistry, Part B, pp. 167-170. Elsevier, Amsterdam. FLORKIN M. & STOTZ E. H. (1975b) Comprehensive Biochemistry, Part B, pp. 172-173. Elsevier, Amsterdam. GREENGARDO., SAHIBM. K. & KNOX W. E. (1970) Developmental formation and distribution of arginase in rat tissues. Archs Biochem. Biophys. 137, 477~,82. GREENSTEINJ. P. & THOMPSONJ. W. (1943) Range in activity of several enzymes in normal and neoplastic tissues of mice. J. natn. Cancer Inst. 4, 275-281. HIRSH-KOLB H. & GREENBERG D. M. (1968) Molecular characteristics of rat liver arginase. J. biol. Chem. 243, 6123-6129. KOCHAKIANC. D. (1945) The effect of dose and nutritive state on kidney arginase after steroid stimulation. J. biol. Chem. 161, 115-125. KONARSKA L. & TOMASZEWSKI L. (1975a) Studies on
L-arginase of the small intestine. I. Topographical distribution and some properties of the small intestine L-arginase in the rat. Biochem. Med. 14, 250-262. KONARSKA L. & TOMASZEWSKI L. (1975b) Studies on L-arginase of the small intestine. II. Intestinal arginase in young and adult mammals, and its role in maintaining urea body pool. Biochem. Med. 14, 263-273.
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KOSSEL A. & DAKIN H. D. (1904) [)ber die Arginase. Z. physiol. Chem. 41, 321-331. LOWRY O. n., ROSEBROUGHN. J., FARR A. L. 8/: RANDELL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265~275. POREMBSKA Z., BARANCZYKA. ~,~ JACHIMOWICZ J. (1971)
Arginase isoenzymes in liver and kidney of some mammals. Acta biochem, pol. Ig, 77-85. REDDY S. R. R. & CAMPBELLJ. W. (1968) A low molecular weight arginase in the earthworm. Biochim. biophys. Acta 159, 557-560.
0305-0491/78/1115-0545502.00/0
INTESTINAL ARGINASE IN VERTEBRATES AND INVERTEBRATES B. LISOWSKA-MYJAK,L. TOMASZEWSKi a n d L. HRYCKIEWICZ Medical School of Warsaw, Department of Clinical Biochemistry, Institute of Biopharmacy, Regional Veterinary Station Koszalin, 02-097 Warszawa, ul. Banacha 1, Poland
(Received 24 October 1977) Abstract--l. Arginase was found to be present in the intestine in all species of Annelida, Arthropoda and Chordata studied. 2. The activity of intestinal arginase differs from species to species, the differences reaching two orders of magnitude (100 × ). 3. The highest activity of intestinal arginase was observed in the rodents (mouse, rat, hamster). 4. In animals in which the enzyme activity was high or moderately high, arginase activity showed topographical differentiation along the long axis of the intestine.
As a rule, samples of the mucosa for the experiments were taken from the proximal, middle and distal segments of the intestine; the only exceptions were the earthworm, crayfish, cockroach, locust and frog.
INTRODUCTION There are but scant data on the occurrence of arginase activity in intestinal mucosa. So far, the presence of this enzyme was d e m o n s t r a t e d in intestinal m u c o s a of the e a r t h w o r m (Lumbricus terrestris) (Reddy & Campbell 1968), dog (Kossell & Dakin, 1904; K o n a r s k a & Tomaszewski, 1975b), mouse (Greenstein & T h o m p s o n , 1943; Kochakian, 1945), rat (Greengard et al., 1970; K o n a r s k a & Tomaszewski, 1975a), ox (Kossel & Dakin, 1904) and man ( K o n a r s k a & Tomaszewski, 1975b). Arginase was reported to be present in the stomach of the fish Raia clavata (Baret et al., 1966) but its intestine has not been checked for the enzyme activity. This paper presents the results of research on the activity of intestinal arginase in representatives of vertebrates a n d invertebrates.
Homogenates Suspensions of the mucosa or the whole intestine were homogenized in a glass Potter Elvehjem homogenizer, for 1.5 rain at 1900 rev/min. Determination of arginase activity The enzyme activity was determined by measuring the increase in ornithine formed. The homogenates were diluted with 5 m M Tris-HCl buffer, pH 7.5 (to which manganese was not added). The incubation mixture contained (per ml) 20pmoles of arginine, I/tmole of MnCI2, 50/~moles of Tris-HCl buffer pH 9.5, the enzyme preparation, and deionized water to bring the volume to 1 ml. The samples contained 8-304/~g of protein ; in the majority of samples this value ranged from 50 to 150pg. The samples were incubated at 37°C for 15 rain. The reaction was stopped by placing the tubes in a boiling-water bath for 10 rain. The ornithine formed was determined according to Chinard (1952).
MATERIALS AND METHODS
Animals In most animals studied the intestine, isolated immediately after killing of the animal, was cut lengthwise, the intestinal contents were removed and the tissue was linsed and frozen. After 2 7 days, the mucosa was scraped off with a scalpel, weighed and suspended in 4 vol of 1 mM MnCI2 5 mM Tris-HCl buffer. This procedure was applied to herring, salmon, bream, cod, pike-perch, duck, hen, rabbit, coypu, guinea-pig, mink, fox, horse, hog, hippopotamus, ox, sheep, antelope, as well as to human intestine obtained from the Department of Pathological Anatomy. The intestines of the frog, pigeon, mouse, rat and hamster was not subjected to freezing, and the mucosa was suspended in the buffer as soon as possible after killing of the animals. These animals were killed by decapitation. Since it proved impossible to isolate mucosa of small animals (earthworm, crayfish, cockroach, locust) the whole intestine wall was disintegrated, suspended in the buffer and pooled samples were examined. The earthworm's intestine was homogenized immediately after being suspended in the buffer; the intestines of the crayfish, cockroach and locust were frozen; they were homogenized not later than 2 days after.
Protein determination Protein was assyed by the method of Lowry et al. (1951 t, bovine serum albumin being used as a standard. RESULTS The activity of intestinal arginase was determined in 30 animal species, including five invertebrate and 25 vertebrate species. A m o n g the latter, the majority were m a m m a l s (15 species). The results presented in Table 1 are arranged in the order of phylogenetic development of the animals studied. The activities of intestinal arginase in columns 4 a n d 5 are expressed, respectively, as ~moles of o r n i t h i n e / m g of protein per 15 rain and #moles of ornithine/g of mucosa per min. 1. Analysis of the presented data indicates that in the animals studied the enzyme activity shows large variations (by a b o u t two orders of magnitude). In the studied invertebrate species (crayfish, cockroach, locust) the activity is very low, ranging from 0.11 to
545
B. LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
546
Table 1. The activities of intestinal arginase (mean and range) in studied species Number of samples 3
No. I 1
2
3 4 5
6 7 8 9 10
II
12 12a 13 14
15
16 17 18 19 20 21 22 23 24 25
Type: Annelida Lumbricus terrestris (earthworm) Type: Arthropoda Class: Crustacea Astatus (crayfish) Class: Insecta Periplaneta orientalis (cockroach) Acridium migratorius (locust) Type: Chordata Class: Pisces Clupea harenyus (herring) Sahno salar (salmon) Ahramis brama (bream) Gadus callarias (cod) Lucioperca (pike perch) Class: Amphibia Rana esculenta (frog) Class: Ayes Anser domesticus (duck) Callus domesticus (hen) B toiler Columbia lieia (Pigeon) Class : Mammalia Order: Lagomorpha Oryctalogus (rabbit) Order : Rodentia M yopotamus coypus (coypu) Cavia parcellus (guinea-pig) Mus (mouse) Rattus rattus (rat) Cricetus vulyaris (hamster) Order: Carnivora Lutreola lutreola (mink) Canis vulpes (fox) Canis (dog) Order : Perissodactyla Equus (horse) Order: Artiodactyla Sus domestica (hog) Hippopotamus amphibius (hippopotamus)
4
0.19 1.56
1. t9-16.5
4
0.40 0.36-0.45
1.45 1.21 1.73
3
0.16 0.14-0.18 0.11 0.084). 15
1.03 0.97-1.11 0.92 0.66-1.37
1
0.26 0.24-0.29 0.27
1.08 0.84-1.45 1.06
1
0.24
0.92
3
0.59 0.524).63 0.44 0.42-0.48
1.89 1.38-2.29 1.81 1.69-1.92
3
0.17 0.144).20
0.77 0.70-0.83
5
0.10 0.064). 15 0.13 0.09-0.17 0.12 0.114).15 0.69 0.37-1.04
0.80 0.39-1.58 0.72 0.53-0.82 1.41 1.12-1.85 3.53 1.53-4.73
2.23 1.84-2.69
14.41 12.00~ 18.40
1.15 0.89-1.42 1.07 0.91-1.36 9.26 5.48 12.13 5.49 3.72-8.40 7.61 6.12-9.89
4.69 3.10-5.73 5.10 3.22-8.41 100.43 76.45-139.00 43.99 36.22-53.31 84.47 63.80--129.58
0.38 0.22-0.50 0.29 0.21-0.35 0.34 0.12-0.51
1.36 0.88-1.66 1.65 1.35-2.52 3.30 1.00-4.90
0.07 0.06-0.07
0.40 0.38-0.42
3
3
3
3 3 4
3 4 5 6 3 4 5 8 5
3
7 1
Activity pM ornithine/mg /~M ornithine/g prot. per 15 min tissue per 1 min 4 5
0.63 0.53-0.76 0.06
4.51 3.94-5.25 0.23
(continued)
Intestinal arginase in vertebrates and invertebrates
547
Table 1. contd.
No. 1
2
26
Bos taurus
Number of samples 3 6
(oxt 27
Ovis
2
(sheep) 28 29
!
Antelope
(antelope) Order: Primates Homo sapiens
20
(many)
0.40/~moles of ornithine/mg of protein per 15 min. In the lowest vertebrates studied (salmon, bream, cod, pike-perch, herring) the activity is also very low, practically the same as in amphibians (frog) and birds (duck, hen, pigeon). In this group, rather low activity was observed only in the cod and pike-perch (0.44-0.59#moles of ornithine/mg of protein per 15 min). In the frog, hen and duck the activity was very low (0.10-0.17 pmoles of ornithine/mg of protein per 15min), whereas in the pigeon the activity was almost five times as high. Increased arginase activity was observed only in some of the mammals, namely the rabbit, coypu and guinea-pig. It was by a factor of 5-10 higher than the activity in animals at the lower grades of phylogenetic development. On the other hand, distinctly higher arginase activity was observed in some of the rodents, i.e. in the mouse, rat and hamster. The average enzyme activity in Carnivora (fox, mink) is distinctly lower than in rodents. In the species of higher animals the average enzyme activity is low, not exceeding 0.17 pmoles of ornithine/mg of protein per 15 min, except in the Artiodactylous hog and in man, which show moderate activity. 2. Comparison of the activity expressed in pmoles of ornithine/mg of protein per 15min with that expressed as #moles of ornithine/g of mucosa per 1 min, shows that the patterns obtained are very similar. The alternative form of expressing the results does not provide new information as compared with the activity calculated per 1 mg of protein. Our results are at variance with those presented by Dixon & Webb (1964). We found rather high activity of intestinal arginase in the rabbit, guinea-pig and rat, whereas in mouse intestine the observed enzyme activity was not as high as that reported by Dixon & Webb (1964). 3. The results discussed above were average values of arginase activity in the intestines of the animals studied. However, we have also observed differences dependent on the intestinal segment in which the activity was determined. The activity of arginase presented in Table 2 for each animal relates to three segments of the intestine: the proximal, middle and distal. In lower species, in which the average activity is very low or rather low, the activity is the same over the whole length of the intestine (herring, salmon, bream, pike-perch, duck, hen). Beginning with the pigeon, there is a distinct differentiation of
Activity #M ornithine/mg pM ornithine/g prot. per 15 sec tissue per 1 see 4 5 0.12 0.08q).23 0.17 0.12~3.21 0.09
0.86 0.44- 2.14 1.18 0.7(~1.65 0.44
0.38 0.244).52
1.09 0.56 1.62
arginase activity with the topographical location of the segment studied. As a rule, the activity is highest in the segment proximal to the stomach (duodenum), than it decreases, the steepness of the gradient differing from species to species (Fig. 1). This phenomenon is observed in all the species studied up to the level of the hippopotamus, and is observed only in those species in which the arginase activity is high or moderate. In herbivorous animals this topographical differentiation was not observed. 4. The results obtained for the earthworm and the snail call for some additional comments. We have performed at least 10 separate measurements of the intestinal arginase activity in the earthworm, under a variety of conditions: immediately after collecting the animals and on animals kept for differing time periods at the laboratory. The results were highly divergent: from 0.19 to 1.56 #moles of ornithine/mg of protein per 15 min. The earthworms studied were not kept under the same conditions: some of them were kept for a few days in the laboratory, and a part were killed within a short time after digging them out from deeper layers of the soil. These conditions had a distinct effect on the arginase activity: the earthworms kept in the laboratory (fasted) showed an activity of 1.14-1.56/~moles of ornithine/mg of protein per 15 min, which corresponds to 764-990#moles of ornithine/g of mucosa per hr, whereas in the freshly collected specimens the activity ranged from 0.043 to 0.42 #moles of ornithine/mg of protein per 15 min, which corresponds to 18.6-172/~moles of ornithine/g of mucosa per hr. Our observations are in agreement with the similarly expressed data of Campbell (1970a). The activity of intestinal arginase in the snail Helix pomatia is not included in table due to the difficulty in separating the intestine from the liver in this organism. No differences in intestinal arginase activity were observed between the common household hen and the broiler chicken.
DISCUSSION
The activity of intestinal arginase was studied in 30 animal species belonging to the types Annelida, Arthropoda and Chordata. Analysis of the data presented in Table 1 shows large variations of the enzyme
B. LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
548
Table 2. The topographical differentiation of arginase activity along the long axis of the intestine in studied species. Parts of intestine: I, postventricular part; II, middle part; III, distal part; IV, colon Arginase activity
No.
Species
1 Type: Annelida Lumbricus terrestris 2 Type: Arthropoda Class: Crustacea Astacus 3 Class: Insecta Periplaneta orientalis 4 Acridium migratorius 5 Type: Chordata Class : Pisces Clupea harengus 6 Salmo salar 7 Abramis brama 8 Gadus callarias 9 Lucioperca 10 Class: Amphibia Rana esculenta 11 Class : Ayes A riser domesticus 12 Gallus domesticus 12 Broiler 13 Columbia livia 14 Class: Mammalia Order: Lagomorpha Oryctaloous 15 Order: Rodentia Myopotamus coypus 16 Caz;ia parcellus 17 Mus 18 Rattus rattus 19 Cricetus vulgaris 20 Order : Carnivora Lutreola lutreola 21 Canis vulpes 22 Canis 23 Order: Perissodactyla Equus 24 Order : Artiodactyla Sus domestica 25 Hippopotamus amphibius 26 Bos taurus 27 Ovis 28 Antelope 29 Order : Primates Homo sapiens
~M ornithine/mg prot. per 15 min Parts of intestine I I1 tll IV
~M ornithine/g tissue per 1 min Parts of intestine i II III IV
Whole intestine 0.19 1.56
Whole intestine 1.19-16.50
Whole intestine 0.40
Whole intestine 1.45
Whole intestine 0.16 Whole intestine 0.11
Whole intestine 1.03 Whole intestine 0.92
0.27 0.24 0.28 0.59 0.42
0.25 0.21 0.21 0.52 0.46
1.25 0.64 1.26 2.56 1.83
0.26 0.36 -0.66 -
0.97 1.18 0.57 1.84 1.78
0.81 1.36 -1.47
Whole intestine 0.77
Whole intestine 0.17
0.94 0.84 1.38 5.16
0.78 0.75 1.20 3.60
0.48 0.57 0.99 0.63
17.25
14.18
12.39
9.95
6.85 10.68 137.73 55.70 118.80
2.35 6.96 139.89 68.45 98.35
0.51 86.53 22.48 75.30
-37.55 10.70 9.27
4.90
2.01 3.68 4.00
1.44 1.05 1.00
0.64 0.09
0.03
0.67
0.57
0.27
0.16
0.49
0.12
-
9.63
2.88
0.67
0. 30 0.09 0.13 O. I 1
0.04 0.07 0.12 0.05
0.38
0.10 0.11 O.12
0.13 0.54 0.92 0.61
0.60 0.67 0.48
0.19 0.30 0.50 0.23
0.24
0.20
0.94
0.54
O.13 O. 15 0.13 1.14
0.10 O. 12 0.10 0.77
0.09 O. 12 0.09 0.21
2.62
2.18
1.93
1.58 1.94 9.39 6.13 8.16
0.72 1.51 17.67 7.23 14.21
0.12 7.33 2.02 6.26 0.41 0.32 0.12
0.23 0.03
0.51
0.50 0.55 0.40
O.10
0.09
0.06
1.40 0.10 -~ 0.70
activity in these types of animals; four groups may be discerned: 1. Animals with very low arginase activity, up to 0.20/~moles of ornithine/mg of protein per 15 min; this group comprises both phylogenetically lower and higher animals: cockroach, locust, frog, duck, domestic hen, broiler chicken, horse, hippopotamus, ox, sheep and antelope. 2. Animals with rather low activity: 0.220.60pmoles of ornithine/mg of protein per 15min: crayfish, herring, salmon, bream, cod, pike-perch, mink, fox, dog and man. 3. Animals with moderate activity; 0.63-1.15pmoles of ornithine/mg of protein per 15rain: pigeon, coypu, guinea-pig, hog.
k~
1.90 0.99 0.77 1.79
1.80
4. Animals with high activity: 1.15-9.26/~moles of ornithine/mg of protein per 15 min : rabbit, rat, hamster, mouse. We are well aware of arbitrary criteria of this division. However, large differences in arginase activity (even by two orders of magnitude) seemed to justify our choice. The group with the highest activity is most distinctly separated from the others, which is clearly evident from Fig. 2. There is a smooth transition between average values of the enzyme activity for the particular species, a small gradient of the decrease or increase in activity being observed except for the rabbit, rat, hamster and mouse (Fig. 2). The very low or rather low activity is found both in lower and higher animals. Thus, it seems possible to sup-
Intestinal arginase in vertebrates and invertebrates
549
18
14 13 12 II
I0
._c E
9 8
tO
A
7 i:x
\ BA
6
o
% ¢:
;:L
2.6 25 24 2:3 22 21 20 19 1.8 1.7 1,6 1,5 1,4 13 1,2 I.I 1.0 0.9 08 0.7 0.6 05 0,4 0:3 0.2 O I 0
AA
A
A
IA
Ax A +A
I
K
TIT
IK
Fig. 1. The topographical differentiation of arginase activity in different parts of intestine: I, postventricular part; II, middle part; III, distal part; IV, colon. Studied species: mouse OA, hamster OA, rat /XA, rabbit &A, guinea-pig DA, coypu IA, hog xA, pigeon +A, mink O, fox 0, man A, dog &, cod D, pike-perch x, salmon +, bream OB, herring liB, duck, hen, hippopotamus, ox, sheep, antelope 1.
pose that higher animals had a higher activity of intestinal arginase, which subsequently they had lost or that the rodents selectively elaborated, for unknown reasons, high activities. Moreover, it should be noted that the activity of intestinal arginase showed topographical differences. The results presented permit us to discern in this respect two groups of animals: (1)--those in which arginase activity is the same over the whole length of the intestine, irrespective of the level of the enzyme activity (herring, salmon, bream, cod, pike-perch, duck, domestic hen, broiler chicken, horse, hippopotamus, ox, sheep, antelope); (II)----those in which there is a distinct topographical differentiation of the activity (Fig. 1) which is the
highest in jejunum and decreases gradually, with differing gradients, toward the distal end of the intestine (pigeon, rabbit, coypu, guinea-pig, mouse, rat, hamster, dog, fox, mink, hog, man). It should be noted that in the present study, only adult animals were used. Arginase activity is also known to depend on the stage of ontogenetic development (rat, man, Fasciola hepatica). The activity of intestinal arginase in the rat up to the second week of life is low (of the order of 1-2/~moles of ornithine/mg of protein per 15 min). An increase in the activity is observed between the second and fourth week, adult values being reached at 5 weeks of life (Konarska & Tomaszewski, 1975a). Similarly, it was found that the activity of intestinal arginase is lower
550
B, LISOWSKA-MYJAK,L. TOMASZEWSKIand L. HRYCKIEWICZ
13
12
10
90
8.0 .E E
70
o.
60 Q. 50
o
40
30 >7 >
n-
28 26 24 22 20 18 16 14 12 ~ o
I0 09 08 0.7 06 0.5 04 0.3 02 OI
1
-
-~.c
-
o
-
ocn
o
u,~
o ~ ¢: o
..~
"~EE
iE
00~
m
°
o~
i1)
, , , , o, o4 1
*~
oo
~o
,
Fig. 2. The comparison of the means of activities of intestinal arginase in species arranged in phylogenetic development.
in children in the first year of life than in adults (Konarska & Tomaszewski, 1975a). On the other hand, in Fasciola hepatica no arginase activity was found in the eggs, miracidium and metacercaria, but was found in the youthful, 15-day-old form of the fluke (Campbell, 1970a). The studies performed so far on the urea cycle limited mainly to two organs: liver and kidney (Hirsh-Kolb & Greenberg, 1968; Porembska et al.,
1971). The functioning of the full urea cycle is most efficient in liver. The discovery of inborn errors of metabolism and some other clinical observations pointed to the possibility of extrahepatal urea formation (Konarska & Tomaszewski, 1975a). Florkin & Stotz (1975a) considers that ureogenesis in various animal species plays a role in protection against changes in osmotic pressure of the environment. Thus, it would follow that the efficiency of the
Intestinal arginase in vertebrates and invertebrates urea cycle in salt-watcr fish should be greater than in freshwater ones. In our experiments both salt-water species (cod, herring) and species living in brackish water (salmon, bream, pike-perch) were studied but the results obtained showed no distinct differences between these groups, and thus did not confirm Florkin & Stotz's view, at least as concerns intestinal arginase. At this stage of our work we have no data on the enzyme activity in the intestine of freshwater fish, and we are unable to characterize the intestinal arginase with respect to different osmotic conditions. In analysing the activity of intestinal arginase in various animal species one should not disregard the effect of feeding. The fasted earthworm is known to have higher enzyme activity than the fed animal (Campbell, 1970b). Our experiments included another example of wide differences in feeding of the same species: the domestic hen and artificially fed broiler chick. These differences, however, did not affect either the level of the activity or its topographical distribution. Thus it seems that in the hen activity of intestinal arginase follows a species-dependent pattern. Topographical differentiation of the intestinal arginase activity in animals with moderate and high enzyme activity (Fig. 1) is of considerable interest. It is not clear whether this differentiation is a speciesdependent property, or is a secondary trait formed due to differences in the type of feeding. However, there are no data which would permit elucidation of this question. The observed differences in the intestinal arginase activity in various animal species are not an isolated phenomenon; also the hepatic arginase is known to show species-dependent differences (Campbell, 1970c), e.g. the activity expressed as units/gram of wet tissue weight was 1000 in the frog, 658 in lion, 458 in ox and 388 in rat. Similar differences in the activity of hepatic arginase were found in birds, e.g. 220 units in the kingfisher, but no activity at all in pigeon. The presented data permit to state that the differences in the arginase activity in various animals are speciesdependent, genetically determined traits. It seems rather striking that no arginase activity was found in pigeon liver, whereas in pigeon intestine the activity reached moderate values. In considering the results obtained one should take into account differences in the functioning of arginase at the particular level of phylogenetic development. Arginase lowers the efficiency of arginine biosynthesis which supplies the body with this exogenous amino acid; it leads also to formation of other metabolites (glutamic acid, proline, polyamines) and of urea indispensable for osmotic regulation. Our work contains several new observations, which lead to a number of unresolved questions. First, is high activity of intestinal arginase an isolated phenomenon, or is it accompanied by an increase in the activity of other enzymes of the urea cycle and increase in its overall efficiency? As concerns higher animals, there are no experimental data permitting us to answer this question. If it is an isolated phenomenon, independent of the activity of other enzymes of the urea cycle, one could expect that intestinal arginase plays a special role in the body of these animals. It seems impossible to reject the assumption
551
that the role of intestinal arginase is not limited to detoxication of ammonia, but would also include formation of urea by by-passing ammonia binding. We are unable to explain why intestinal arginase activity is high in rodents and the rabbit (La#omorpha). Is their metabolism so widely different that it requires additional formation of urea in the intestinal mucosa? F r o m observations of pathological cases we know that in man the increase in the activity of intestinal arginase may lead to an increase in the extracellular pool of urea (Konarska & Tomaszewski, 1975a). The aim of our work was a very modest and limited one: to inquire into the activity of arginase in phylogenetic development, in a tissue not typical for ureogenesis, i.e. the intestine. We have demonstrated very wide differences in the enzyme activity and its topographical differentiation. These facts point to a number of unresolved problems, which may shed new light on the alternative role of arginase as a separate source of urea formation, a function not related to the role of urea cycle in the metabolism of ammonia.
REFERENCES BARET R., MOURGUE• PELLEGRINJ. (1966) Sur l'existence de deux activit6s arginasiques dans les tissus hepatique, r6nal et stomacal de la raie clout6e. C. R. Sdanc. Soe. biol. 160, 1796-1800. CAMPBELL J. W. (1970a) Comparative Biochemistry oJ Nitrogen Metabolism. Vol. 1, The Invertebrates, p. 84, Academic Press, New York. CAMPBELL J. W. (1970b) Comparative Biochemistry oJ Nitrogen Metabolism. Vol 1, The Invertebrates, p. 256. Academic Press, New York. CAMPBELL J. W. (1970c) Comparative Biochemistry oJ Nitrooen Metabolism. Vol. 2, The vertebrates, p. 750. Academic Press, New York. CmNARD F. P. (1952) Photometric estimation of proline and ornithine J. biol. Chem. 199, 91-95. DIXON M. & WEBB E. (1964) Enzymes pp. 636-645. Longmans, Green, London. FLORKIN M. & STOTZ E. H. (1975a) Comprehensive Biochemistry, Part B, pp. 167-170. Elsevier, Amsterdam. FLORKIN M. & STOTZ E. H. (1975b) Comprehensive Biochemistry, Part B, pp. 172-173. Elsevier, Amsterdam. GREENGARDO., SAHIBM. K. & KNOX W. E. (1970) Developmental formation and distribution of arginase in rat tissues. Archs Biochem. Biophys. 137, 477~,82. GREENSTEINJ. P. & THOMPSONJ. W. (1943) Range in activity of several enzymes in normal and neoplastic tissues of mice. J. natn. Cancer Inst. 4, 275-281. HIRSH-KOLB H. & GREENBERG D. M. (1968) Molecular characteristics of rat liver arginase. J. biol. Chem. 243, 6123-6129. KOCHAKIANC. D. (1945) The effect of dose and nutritive state on kidney arginase after steroid stimulation. J. biol. Chem. 161, 115-125. KONARSKA L. & TOMASZEWSKI L. (1975a) Studies on
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KOSSEL A. & DAKIN H. D. (1904) [)ber die Arginase. Z. physiol. Chem. 41, 321-331. LOWRY O. n., ROSEBROUGHN. J., FARR A. L. 8/: RANDELL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265~275. POREMBSKA Z., BARANCZYKA. ~,~ JACHIMOWICZ J. (1971)
Arginase isoenzymes in liver and kidney of some mammals. Acta biochem, pol. Ig, 77-85. REDDY S. R. R. & CAMPBELLJ. W. (1968) A low molecular weight arginase in the earthworm. Biochim. biophys. Acta 159, 557-560.