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Formation of argininosuccinate in cattle erythrocytes
Comp. Biochem. Physiol., 1973, Vol. 44B, pp. 939 to 941. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION F O R M A T I O N OF A R G I N I N O S U C C I N A T E IN CATTLE ERYTHROCYTES W. BAREY* and J. H A R M E Y E R Institute of Physiology, School of Veterinary Medicine, 3 Hannover, Bischofsholer Damm 15, Germany
(Recdved 11 July 1972) A b s t r a c t - - T h e presence of argininosuccinate synthetase activity was demonstrated in cattle erythrocytes but not in goats. The activity per g fresh weight, however, was less than 1 per cent compared to that found in the liver. The physiological role of this urea cycle enzyme in the blood cells cannot as yet be assessed. INTRODUCTION IN PREVIOUS studies, measurable amounts of argininosuccinate were detected in blood cells of cattle whereas it was absent in goats (Barey, 1970). In order to evaluate the origin of this urea cycle intermediate studies on argininosuccinate synthetase (E.C. 6.3.4.5) activity in blood cells of these two ruminant species were conducted. MATERIALS AND METHODS The corpuscular elements of fresh blood from cattle and goats were separated by centrifugation and hemolyzed at 5°C. Argininosuccinate synthetase activity was assayed following essentially the method of Brown & Cohen (1959) using ureido-14C-e-citrulline (NEN Corp., Frankfurt, Germany). Appropriate cell-free blanks, heated controls, as well as parallels of liver homogenates were included, l~C-Argininosuccinate was separated by ion exchange chromatography and determined by a Packard Tricarb flow through assembly in connection with an automatic amino acid analyser. RESULTS T h e results are summarized in Table 1. Argininosuccinate synthetase activity could be demonstrated in blood cells of cattle but not in goats. T h e amount of argininosuccinate increased with the time of incubation. Slightly higher amounts of argininosuccinate were detected by the colorimetric ninhydrin method as compared to the radioactivity measurements. No labelled argininosuccinate, arginine or urea could be detected in goat blood * Supported by a grant from the West German Humboldt Foundation. 939
940
W . BAREY AND J. HARMEYER
TABLE 1--FORMATION OF 14C-ARGININOSUCCINATE BY CATTLE BLOOD CELLS DURING INCUBATION WITH UREIDO-14C-L-CITRULLINE //xmole/g/ (FIVE EXPERIMENTS)
No. of Min
Blood cells of cattle Bovine liver
30
90
150
0'08 + 0"03
0-22 _+0"05
0-36 + 0'09
24"77
1"1/zmole of urea during 60 min
cell preparations indicating the absence of agininosuccinase activity in the preparation of this ruminant species. The argininosuccinate synthetase activity in blood cells of cattle was about 1 per cent of that found in the bovine liver (Table 1). In a previous paper (Barey & Hill, 1970) it was shown that blood cells of cattle also exhibit remarkable arginase activity being of the same order of magnitude as that found in the bovine liver, while not being present in goats. DISCUSSION At present it is difficult to understand the physiological significance of argininosuccinate synthetase activity in cattle blood cells. The product of its reaction was first isolated in 1953 by Ratner& Petrack from mammalian liver preparations as an intermediate metabolite of urea formation. Argininosuccinate is formed by the action of argininosuccinate synthetase which combines citrulline and aspartic acid at the expense of an ATP. The condensing reaction is regarded as the rate limiting step in urea biosynthesis (Cohen & Brown, 1960). In subsequent investigations argininosuccinate synthetase activity was detected in various bacterial species, plants and vertebrate tissues. In mammals the localization of the enzyme is not organ specific as has been found in kidney, muscle, pancreas and brain (Ratner, 1962). Argininosuccinate may also be regarded as a precursor in the synthesis of arginine and creatine. Since argininosuccinase activity (E.C. 4.3.2.1) is not present in blood cells argininosuccinate possibly leaves the blood cells and enters the plasma. This is supported by the observation that neither x4-C-arginine nor 14C-urea could be detected in cattle blood preparations. With respect to the high arginase activity in cattle blood cells the precise physiological role of this enzyme remains obscure. It is well known, however, that ruminants show several unique properties concerning protein digestion and ammonia metabolism. When the animals receive high urea supplemented diets a considerable amount of ammonia is formed in the tureen and diffuses partly into the blood stream. One may consider that argininosuccinate synthetase participates in the detoxification of ammonia and the urea formation. REFERENCES BAREYW. (1970) Comparative investigations of the concentration of free amino acids in the plasma and blood cells of cattle and goats. Acta Physiol. Pol. 21, 801-809.
ARGININOSUCCINATE IN CATTLE ERYTHROCYTES
941
BAREY W. & HILL H. (1970) Arginine metabolism in the blood cells of cattle and goats. Q. jY. exp. Physiol. 55, 64--68. BaOWN G. W. & COHEN P. P. (1959) Comparative biochemistry of urea synthesis--I. Methods for the quantitative assay of urea cycle enzymes in liver. 3. biol. Chem. 234, 1769-1774. COHEN P. P. & BaOWN G. W. (1960) Ammonia metabolism and urea biosynthesis. In Comparative Biochemistry (Edited by FLOnKINM. & MASONH. S.), Vol. II, pp. 161-244. Academic Press, New York. RAT~ZEnS. (1962) Nitrogen transfer from aspartic acid in the formation of amide, amidine, and quanidino groups. In The Enzymes (Edited by BoYEa P. D., LARDyH. & MY~XCK K.), Vol. 6, pp. 495-513. Academic Press, New York. RAT~n S. & Pm~CK B. (1953) Biosynthesis of urea--IV. Further studies on condensation in arginine synthesis from citruUine. 3. biol. Chem. 200, 161-174.
Key Word Index--Argininosuccinate; red blood cells; cattle RBC; goat RBC; synthetase.
SHORT COMMUNICATION F O R M A T I O N OF A R G I N I N O S U C C I N A T E IN CATTLE ERYTHROCYTES W. BAREY* and J. H A R M E Y E R Institute of Physiology, School of Veterinary Medicine, 3 Hannover, Bischofsholer Damm 15, Germany
(Recdved 11 July 1972) A b s t r a c t - - T h e presence of argininosuccinate synthetase activity was demonstrated in cattle erythrocytes but not in goats. The activity per g fresh weight, however, was less than 1 per cent compared to that found in the liver. The physiological role of this urea cycle enzyme in the blood cells cannot as yet be assessed. INTRODUCTION IN PREVIOUS studies, measurable amounts of argininosuccinate were detected in blood cells of cattle whereas it was absent in goats (Barey, 1970). In order to evaluate the origin of this urea cycle intermediate studies on argininosuccinate synthetase (E.C. 6.3.4.5) activity in blood cells of these two ruminant species were conducted. MATERIALS AND METHODS The corpuscular elements of fresh blood from cattle and goats were separated by centrifugation and hemolyzed at 5°C. Argininosuccinate synthetase activity was assayed following essentially the method of Brown & Cohen (1959) using ureido-14C-e-citrulline (NEN Corp., Frankfurt, Germany). Appropriate cell-free blanks, heated controls, as well as parallels of liver homogenates were included, l~C-Argininosuccinate was separated by ion exchange chromatography and determined by a Packard Tricarb flow through assembly in connection with an automatic amino acid analyser. RESULTS T h e results are summarized in Table 1. Argininosuccinate synthetase activity could be demonstrated in blood cells of cattle but not in goats. T h e amount of argininosuccinate increased with the time of incubation. Slightly higher amounts of argininosuccinate were detected by the colorimetric ninhydrin method as compared to the radioactivity measurements. No labelled argininosuccinate, arginine or urea could be detected in goat blood * Supported by a grant from the West German Humboldt Foundation. 939
940
W . BAREY AND J. HARMEYER
TABLE 1--FORMATION OF 14C-ARGININOSUCCINATE BY CATTLE BLOOD CELLS DURING INCUBATION WITH UREIDO-14C-L-CITRULLINE //xmole/g/ (FIVE EXPERIMENTS)
No. of Min
Blood cells of cattle Bovine liver
30
90
150
0'08 + 0"03
0-22 _+0"05
0-36 + 0'09
24"77
1"1/zmole of urea during 60 min
cell preparations indicating the absence of agininosuccinase activity in the preparation of this ruminant species. The argininosuccinate synthetase activity in blood cells of cattle was about 1 per cent of that found in the bovine liver (Table 1). In a previous paper (Barey & Hill, 1970) it was shown that blood cells of cattle also exhibit remarkable arginase activity being of the same order of magnitude as that found in the bovine liver, while not being present in goats. DISCUSSION At present it is difficult to understand the physiological significance of argininosuccinate synthetase activity in cattle blood cells. The product of its reaction was first isolated in 1953 by Ratner& Petrack from mammalian liver preparations as an intermediate metabolite of urea formation. Argininosuccinate is formed by the action of argininosuccinate synthetase which combines citrulline and aspartic acid at the expense of an ATP. The condensing reaction is regarded as the rate limiting step in urea biosynthesis (Cohen & Brown, 1960). In subsequent investigations argininosuccinate synthetase activity was detected in various bacterial species, plants and vertebrate tissues. In mammals the localization of the enzyme is not organ specific as has been found in kidney, muscle, pancreas and brain (Ratner, 1962). Argininosuccinate may also be regarded as a precursor in the synthesis of arginine and creatine. Since argininosuccinase activity (E.C. 4.3.2.1) is not present in blood cells argininosuccinate possibly leaves the blood cells and enters the plasma. This is supported by the observation that neither x4-C-arginine nor 14C-urea could be detected in cattle blood preparations. With respect to the high arginase activity in cattle blood cells the precise physiological role of this enzyme remains obscure. It is well known, however, that ruminants show several unique properties concerning protein digestion and ammonia metabolism. When the animals receive high urea supplemented diets a considerable amount of ammonia is formed in the tureen and diffuses partly into the blood stream. One may consider that argininosuccinate synthetase participates in the detoxification of ammonia and the urea formation. REFERENCES BAREYW. (1970) Comparative investigations of the concentration of free amino acids in the plasma and blood cells of cattle and goats. Acta Physiol. Pol. 21, 801-809.
ARGININOSUCCINATE IN CATTLE ERYTHROCYTES
941
BAREY W. & HILL H. (1970) Arginine metabolism in the blood cells of cattle and goats. Q. jY. exp. Physiol. 55, 64--68. BaOWN G. W. & COHEN P. P. (1959) Comparative biochemistry of urea synthesis--I. Methods for the quantitative assay of urea cycle enzymes in liver. 3. biol. Chem. 234, 1769-1774. COHEN P. P. & BaOWN G. W. (1960) Ammonia metabolism and urea biosynthesis. In Comparative Biochemistry (Edited by FLOnKINM. & MASONH. S.), Vol. II, pp. 161-244. Academic Press, New York. RAT~ZEnS. (1962) Nitrogen transfer from aspartic acid in the formation of amide, amidine, and quanidino groups. In The Enzymes (Edited by BoYEa P. D., LARDyH. & MY~XCK K.), Vol. 6, pp. 495-513. Academic Press, New York. RAT~n S. & Pm~CK B. (1953) Biosynthesis of urea--IV. Further studies on condensation in arginine synthesis from citruUine. 3. biol. Chem. 200, 161-174.
Key Word Index--Argininosuccinate; red blood cells; cattle RBC; goat RBC; synthetase.