- Email: [email protected]
Urea cycle enzymes in tissues (liver, rumen epithelium, heart, kidney, lung and spleen) of sheep (Ovis aries)
Comp. Biochem. Physiol,, Vol. 65B, pp. 693 to 697 © Pergamon Press Ltd 1980. Printed in Great Britain
0305-0491/80/0401-0693502.00/0
UREA CYCLE ENZYMES IN TISSUES (LIVER, RUMEN EPITHELIUM, HEART, KIDNEY, LUNG AND SPLEEN) OF SHEEP (OVIS ARIES) B. EMMANUEL Department of Animal Biology, School of Veterinary Medicine, University of Shiraz, Shiraz, Iran (Received 20 July 1979) Abstract--1. The enzymes of urea cycle were studied in the tissues (liver, rumen epithelium, heart,
kidney, lung and spleen) of sheep (Ovis aries). 2. The complete urea cycle was functional in significant quantities only in the liver. 3. Activities of urea cycle enzymes (carbamyl phosphate synthetase, ornithine transcarbamylase, arginine synthetase and arginase) were much higher in the liver than in other tissues; the enzymic pattern in other tissues tested was the same. 4. It is concluded that the rumen epithelium disposes very little ammonia through the urea cycle. Other mechanisms in addition to glutamate dehydrogenase and glutamine synthetase are expected to exist in this tissue to avoid ammonia toxicity. The rumen epithelium is exposed to very high concentrations of ammonia (5-55 mg NH3-N/100 ml rumen contents).
INTRODUCTION
The degradation of nitrogenous compounds by rumen micro-organisms leads to the production of high levels of ammonia in the rumen (Hungate, 1968; Church, 1971). The entry rates and concentrations of ammonia in the rumen of sheep fed conventional diets respectively were: 14-15g NH3-N/day (Nolan & Leng, 1972; Nolan et al., 1976) and 5-55mg NH3-N/100ml rumen contents (Hungate, 1968; Mathison & Milligan, 1971; Mehrez et al., 1977). Of the total ammonia entering the rumen pool about 40% is absorbed through the rumen wall, 46% is incorporated into microbial protein and 16% flows to the lower part of the digestive tract (Nolan, 1975). Interestingly enough, the levels of ammonia in the blood of ruminants and monogastrics are comparable {Diem & Lentner, 1970; Shin & Efron, 1972; Davidovich et al., 1977a). On the other hand, concentrations of ammonia in the rumen are 50-100 times higher than in portal blood (Bartley et al., 1976; Davidovich et al., 1977b). Therefore, highly efficient mechanisms of ammonia assimilation are expected to be present in the rumen epithelium to prevent ammonia toxicity in this tissue. The occurrence of the following enzymes, utilizing ammonia in the rumen epithelium has been reported: glutamate dehydrogenase (EC 1.4.1.3) (Cooper, 1962; Ide, 1969b; Yavonenko, 1969; Yavonenko et al., 1972), glutamine synthetase (EC 6.3.1.2) (Hoshino et al., 1966; Chalupa et al., 1970; Salem et al., 1973), and transaminases (glutamate-oxalacetate, glutamate-pyruvate, ornithine, tyrosine, tryptophan and phenylalanine (Chalupa et al., 1970; Whanger & Church, 1970; Weeks, 1972, 1974). Data on the presence of urea cycle enzymes in the rumen epithelium is not conclusive Arginase (EC 3.5.3.1) was found in the rumen epithelium of cattle (Martin~i6 & Krvavica, 1964; Kurelec et al., 1968; Whanger & Church, 1970), and goat (Kurelec et al., 1968), and ornithine transcarbamylase (EC 2.1.3.3) in the rumen wall of cattle (Hol-
tenius & Jacobsson, 1966; Harmeyer et al., 1968; Whanger & Church, 1970), and goat (Harmeyer et al., 1968; Ide, 1969a). Krvavica et al. (1964), and Ide (1969a) could not detect any activity of carbamyl phosphate synthetase (EC 2.7.2.5) in the rumen epithelium of cattle and goat while Salem et al. (1973) reported its presence in the rumen epithelium of calves. The activity of argininosuccinate synthetase (EC 6.3.4.5), and argininosuccinase (EC 4.3.2.1), and the complete function of the urea cycle have not been investigated in the rumen epithelium. No work has been carried out on the enzymes of urea cycle in the rumen epithelium of sheep. The present work was aimed to investigate the possibility of the operation of complete urea cycle (the incorporation of ammonia and COz into urea), and the activity of the five enzymes of this cycle in the rumen epithelium of sheep. Other tissues including liver, heart, kidney, lung and spleen were used for comparison. MATERIALS AND METHODS
L-[14C]guanido-arginine (55 mCi/mmol), L-[ ! 4C]ureidocitrulline (62mCi/mmol), and [t4C]sodium bicarbonate (59.9mCi/mmol) were purchased from Radiochemical Centre, Amersham, Bucks, U.K. L-citrulline-HC1,L-ornithine-HC1, L-arginine, N-acetyl glutamic acid, L-aspartic acid, 3-phosphoglyceric acid, glutathione, carbamyl phosphate and urease (type III) were the products of Sigma (London) Chemical Co. Tissue homogenate Tissues (rumen epithelium, liver, heart, kidney, lung and spleen) were obtained from Shiraz city abattoir immediately after slaughter, kept in ice and transferred within 45 rain to the laboratory. Rumen epithelium was stripped from the underlying tissues, washed with physiological saline a few times while stirring (to remove food particles and bacteria), and then blotted. A homogenate of all tissues was made of 1 part tissue and 9 parts of a solution of 0.025 M KH2PO4 (pH 7.8), using a Silverson homogenizer
693
694
B. EMMANt:EC
acid synthesis are located in the mitochondria and cytoplasm, respectively (McGilvery, 1970). However, carbamyl phosphate formed can freely pass the mitochondrial membrane (Natale et al., 1974). Both carbamyl phosphate synthetase and ornithine transcarbamylase are mitochondrial enzymes. The work of Ide (1969a) showing 14 times higher ornithine transcarbamylase specific activity in the mitochondrial fraction than whole rumen mucosa may indirectly indicate the presence of carbamyl phosphate synthetase in the rumen epithelial mitochondria. Regardless in what compartment carbamyl phosphate is formed, it can be further utilized for citrulline synthesis in the mitochondria. The activity of liver carbamyl phosphate synthetase was about 20 times higher than the rumen epithelium. Other tissues showed similar activities. Sheep liver activity was higher than that of cattle (5.79 units/g wet wt) (Krvavica et al., 1964), but lower than that of goat (487 557 units) (Brown & Cohen, 1960; Ide, 1969a). The activity of liver ornithine transcarbamylase was high (1877 units), about 55 82 times the values found in other tissues. The pattern in different tissues was similar to the data of Holtenius & Jacobsson (1966) performed on cattle tissues. Other workers (Krvavica et al., 1964; Whanger & Church, 1970) showed lower values (2(~110 units) in this species. On the other hand, activities reported for goat liver were 19,250-24,000 units (Cohen & Brown, 1960; Ide, 1969a). The activity in rat liver tested here was 5920 units/g wet wt, and the enzyme was completely dependent on ornithine and carbamyl phosphate. The activity in the rumen epithelium was respectively 2.4 and 4 times the values found in the goat (Ide, 1969a), and cattle (Krvavica et al., 1964; Whanger & Church, 1970). Activities of arginine synthetase which utilizes two consecutive reactions (argininosuccinate synthetase and argininosuccinase) were 50 and 14 units for the liver and rumen epithelium, respectively. The values of argininosuccinate synthetase found in the liver of goat and ox were 75 and 92 units, respectively (Brown & Cohen, 1960). RESULTS AND DISCUSSION Arginase activity in the rumen epithelium was 26.5 The results on enzyme activities are tabulated in units/g wet wt, The values reported for the rumen Table 1. Some activity (3 units/g wet wt) of carbamyl epithelium of cattle were 30-205 units (Martin6ic" & phosphate synthetase in the rumen epithelium was Krvavica, 1964; Whanger & Church, 1970). The value found. This finding agrees with the work of Salem et of 50.7 units found in the kidney agrees with that of al. (1973) performed on calves, but contrasts the cattle (Martin6ic' & Krvavica, 1964). The data on the results of Krvavica et al. (1964), and Ide (1969a) who spleen, heart, lung and kidney was of the same order. observed no activity in the rumen epithelium of the The liver had the highest activity which is in accord cattle and goats. In the present studies, whole rumen with the data on cattle liver (Martin6ic' & Krvavica, 1964). On the other hand, this value is much lower epithelial homogenate was used whereas, those authors utilized cell-free extracts. Previous work than that reported in earlier studies in goat liver (unpublished data) showed that 3-hydroxybutyrate (Brown & Cohen, 1960). From the literature data predehydrogenase (EC 1.1.1.30), a mitochondrial enzyme, sented here, it appears that there are species differis tightly bound to the cell particles in the rumen ences in ruminants with respect to enzyme activities epithelium, and is only partially released by liquid of urea cycle. The complete urea cycle which utilizes all five nitrogen and sonication treatments. Therefore, it is likely that in cell-free extract preparation, carbamyl enzymes was shown to be functional significantly only phosphate synthetase was not present. Carbamyl in the liver (700-850 times higher than other tissues). phosphate is needed not only for urea synthesis, but The activity (30 units/g wet wt) was lower than the also for nucleic acids formation. Thus, it is logical to activity of each enzyme separately. This is expected expect the presence of this enzyme in the rumen epith- since carbamyl phosphate synthetase, argininosuccielium which is metabolically an active tissue. The nate synthetase and argininosuccinase are rate limitcarbamyl phosphate synthetases for urea and nucleic ing steps in the urea cycle as shown in different
at a moderate speed. Whole homogenate was used for urea cycle enzyme assays. Arginase, arginine synthetase and complete urea cycle were assayed essentially the same as described previously (Emmanuel & Gilanpour, 1978) with very little modifications. The arginase (EC 3.5.3.1) assay consisted of 100 pmols of Tris buffer (pH 8.0), 50 #mols sodium glycinate (pH 9.5), 2.5 #mols L-arginine(pH 9.5), 0.5/aCi e-[14C]guanido-arginine, 0.5/~mols MnCIz, and 100 #1 tissue homogenate. The arginine synthetase (argininosuccinate synthetase plus argininosuccinase) assay contained 100pmols Trisbuffer (pH 8.0), 30 #mols MgSO4, 10 #tools L-aspartic acid, 5 pmols ATP (pH 7.0), 5 pmols L-citrulline-HCl, 0.5/xCi L-[14C]ureido-citrulline and 200 #1 tissue homogenate. The reaction mixture for the complete urea cycle assay was composed of 100 #tools Tris buffer (pH 8.0), 10 pmols ATP (pH 7.0), 10pmols L-ornithine-HC1, 10pmols Nacetyl glutamic acid, 30pmols MgSO4, 16 pmols reduced glutathione, 10#mols L-aspartic acid, l#mol MnC12, 5 pmols 3-phosphoric acid (to regenerate ATP), 10 pmols NaHI4CO3 (in 1 N NaOH to prevent escape of CO2). The reaction mixture was then made to pH 7.5 by adding 70 pl of 1N HCI, and 10pmols of NH4HCO3 and 100pl of tissue homogenate were added. The subsequent procedures up to the counting of the radioactivity were the same as described previously (Emmanuel & Gilanpour, 1978). The ornithine transcarbamylase (EC 2.1.3.3) assay contained 300,umols Tris-buffer (pH 8.0), 10pmols L-ornithine HCI, 10/~mols carbamyl phosphate, 2 mg urease and 100 pl of tissue homogenate in a total volume of 3 ml. The reaction mixture was incubated in a shaking water bath at 37°C for 30min. At the end of the incubation time, the reaction was terminated by adding 2 rnl of 15% metaphosphoric acid. The determination of the product (citrulline) formed was made as described by Archibald (1944). The carbamyl phosphate synthetase (EC 2.7.2.5) assay consisted of 300 #mols Tris buffer (pH 8.0), 25/~mols ATP (pH 7.0), 20 pmols L-ornithine-HCl, 25/tmols N-acetyl glutamic acid (pH 7.0), 16 pmols reduced glutathione (pH 7.0), 30 pmols MgSO4, 12.5,umols 3-phosphoglyceric acid (pH 7.0), 50 pmols NH4HCO3 and 250 pl of tissue homogenate in a total volume of 3ml. The citrulline formed was measured as described above. Since all tissues tested had an active ornithine transcarbamylase, the addition of this enzyme commercial was unneccessary.
Urea cycle enzymes in sheep tissues
695
Table 1. Activities (units*/g wet wt) of urea cycle enzymes in the sheep (Ovis aries) tissues Enzyme Tissue Liver
Mean + SD
Carbamyl phosphate synthetase 60.4 75.4 47.2 --61.0 14.1
Spleen
7.10 6.91 7.62 --
Mean + SD Lung
Mean _ SD Heart
Mean ___SD Kidney
Mean + SD Rumen epithelium
Mean + SD
Ornithine transcarbamylase 1524 1646 1783 1890 2540 1877 396
Arginine** synthetase 56.7 52.2 50.0 42.4 48.3 49.9 5.25
Arginase 648 613 545 310 470 517 134
Completer urea cycle 27.4 14.6 39.8 22.7 44.3 29.8 12.1
32.5 24.4 32.6 34.4 47.5 34.3 8.34
89.3 13.2 46.7 58.4 81.9 57.9 30.4
0.048 0.039 0.043
7.21 0.36
30.7 30.5 32.4 37.0 24.8 31.1 4.38
6.90 8.38 4.57 --6.62 1.91
30.5 32.4 48.6 25.7 22.9 32.0 10.03
36.0 23.7 28.7 31.2 34.8 30.9 4.95
54.7 25.4 49.2 73.0 75.8 55.6 20.4
0.030 0.048 0.037
5.70 6.10 4.57 --5.46 0.78
27.4 36.2 32.4 23.8 30.4 30.0 4.71
37.0 31.7 20.6 39.4 40.2 33.8 8.08
50.6 26.7 48.5 57.0 78.0 52.2 18.4
0.031 0.037 0.033
5.81 6.10 6.86 --6.25 0.57
24.4 22.3 24.8 17.1 26.7 23.1 3.68
35.7 26.6 36.0 35.6 50.1 36.8 8.42
27.2 49.7 45.6 52.9 78.2 50.7 18.3
0.031 0.045 0.028 ---0.035 0.009
3.00 3.81 2.29 --3.03 0.79
33.3 36.2 28.6 34.3 36.2 33.7 3.12
16.9 11.1 14.5 12.9 13.1 13.7 2.16
18.2 30.6 28.9 19.5 35.1 26.5 7.23
0.038 0.032 0.035 --0.035 0.003
-0.043 0.005
0.038 0.009
-0.034 0.003
* Unit of enzyme activity is expressed as 1 ,umol product formed/hr at 37°C. ** Arginine synthetase consists of the activities of argininosuccinate synthetase plus argininosuccinase. 1"Complete urea cycle utilizes all the reacions leading from ammonia and CO2 to the formation of urea. species (Brown & Cohen, 1960). The complete urea cycle developed (Emmanuel & Gilanpour, 1978) is very sensitive. The reaction was completely dependent on ATP, N-acetyl glutamic acid, magnesium and tissue homogenate. The activity was reduced by 70, 84 and 94~o in the absence of ornithine, N H 4 H C O 3 , and aspartic acid, respectively. Maximal activity was shown at p H 7.5. It is i m p o r t a n t to note that the results of arginine synthetase, arginase a n d complete urea cycle assays presented here may have beeVn underestimated. As described in the Materials a n d M e t h o d s section (Emmanuel & Gilanpour, 1978), in these enzyme assays, the product formed ([14C]urea) was measured enzymatically, using commercial urease. The presence of urease c.B.P. 65/4B--H
of bacterial origin which is hypothesized to be diffused from the r u m e n into the epithelial cells has been reported (Abdel R a h m a n & Decker, 1966; H o u p t & Houpt, 1968). Therefore, if urease activity was present in the r u m e n epithelial homogenate of the present work, [14C]urea could be hydrolysed to 14CO2 and a m m o n i a during the incubation period, and the former could have been released after acidification, thus resulting in lower values for those enzymes. Nevertheless, the activities of carbamyl p h o s p h a t e synthetase and ornithine transcarbamylase (which are not affected by the presence of urease) in the r u m e n epithelium were 20 a n d 55 times lower than respective values in the liver. The above a r g u m e n t points to the insignificant function of the complete urea cycle in
696
B. EMMANUEL
this tissue. Chalupa et al. (1970) suggested the presence of trace amounts of five urea cycle enzymes in the rumen wall of the sheep. Under certain dietary regimens, the synthesis of urea cycle enzymes in the rumen epithelium may be induced, therefore, contributing more to ammonia assimilation in this tissue. Sheep fed on soy protein diet had higher activities of transaminases and glutamate dehydrogenase in the rumen epithelium than animals fed on a urea diet (Chalupa et al., 1970). Similarly, rumen epithelium glutamine synthetase activities were higher in calves receiving 50~o u r e a - N diet than 0, or 100°~o urea-N diets (Salem et al., 1973). Adaptation characteristics of NH3-assimilating enzymes on dietary protein have been reported in goat liver (Shimayashi & Yonemura, 1970), and rat liver (Schimke, 1962; Stephen, 1968; Das & Waterlow, 1974). Considering the subject from a different angle, the occurrence of some of the urea cycle enzymes may not necessarily point on the operation of the cycle in the tissue under question. As mentioned above, carbamyl phosphate synthetase found in the rumen epithelium may be functional more in nucleic acids than urea synthesis. Hameyer et al. (1968) suggested that urease, arginase and ornithine transcarbamylase operate cooperatively in the rumen epithelium in recirculation of nitrogen compounds between the rumen contents and the blood. Similar to urea cycle enzymes, the activities of transaminases and glutamate dehydrogenase were also lower in the rumen epithelium than in the liver (Whanger & Church, 1970; Salem et al., 1973). Considering this, and the facts that the liver is twice as heavy as the rumen epithelium (Hamada et al., 1976), and ammonia levels in the rumen are markedly higher than in the blood (Bartley et al., 1976), it appears that the above mentioned NH3-assimilating mechanisms are not efficient enough to explain ammonia detoxication in the rumen epithelium. Rumen epithelium is metabolically an active tissue; it is composed of cells containing numerous mitochondria, rough endoplasmic reticulum, Golgi apparatus and large nuclei with prominent nucleoli (Steven & Marshall, 1970). Perhaps the rumen has developed sophisticated mechanisms for ammonia disposal in order to avoid ammonia toxicity. Other mechanisms of NH3-fixation which have not been referred to in the text are asparagine synthetase and reductive amination of keto-acids (such as pyruvate to alanine, and glyoxylate to glycine which occur in bacteria (Cohen & Brown, 1960)). Branched-chain amino acids, phenylalanine and tryptophan in rumen micro-organisms are synthesized through carboxylation of respective keto-acids and then amination (Allison, 1970). Van der Horst (1961) has reported the presence of keto-acids including 2-ketoisovalerate, 2-ketoisocaproate, 2-keto-3-methylvalerate, hydroxypyruvate and glyoxylate in rumen contents. These keto acids may be transaminated or reductively aminated in the rumen epithelium. Further work is required to verify other mechanisms of NH3-assimilation in the rumen epithelium. It is concluded that little amounts of all five urea cycle enzymes were found in the rumen epithelium, however, the function of complete urea cycle in the rumen epithelium is insignificant as compared to the
liver. Other tissues tested showed similar characteristics of urea cycle enzymes as the rumen epithelium. Acknowledgements--The author wishes to thank Mr T. Vaseghee for his excellent technical assistance. This work was supported by a grant from University of Shiraz Research Council.
R EFERENCES
ABDEL RAHMANS. (~ DECKER P. (1966) Comparative study of urease in the rumen wall and rumen contents. Nature, Lond. 209, 618-619. ALLISON M. J. (1970) Nitrogen metabolism of ruminal micro-organisms. In Physiology of Digestion and Metabolism in the Ruminant (Edited by PHILLIPSON A. T.) pp. 456-473. Oriel Press, Newcastle-upon-Tyne. ARCHIBALD R. M. (1944) Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. J. biol. Chem. 156, 121-142. BARTLEYE. E., DAVIDOVICH A. D., BARR G. W., GRIEFEL G. W., DAYTONA. D., DEYOEC. W. & BECHTLE R. M. (1976) Ammonia toxicity in cattle. I. Rumen and blood changes associated with toxicity and treatment methods. J. Anita. Sci. 43, 835-841. BROWN JR G. W. & COHEN P. P. (1960) Comparative biochemistry of urea synthesis. 3. Activities of urea-cycle enzymes in various higher and lower vertebrates. BiDchem. J. 75, 82-91. CHALUPA W., CLARK J., OPL1GER P. & LAVKER R. (1970) Ammonia metabolism in rumen bacteria and mucosa from sheep fed soy protein or urea. d. Nutr. 100, 161 169. CHURCH D. C. (1971) Digestive Physiology and Nutrition of Ruminants, Vol. 1. pp. 199 223. O.S.U. Book Stores, Corvallis. COHEN P. P. & BROWNJR G. W. (1960) Ammonia metabolism and urea biosynthesis. In Comparative Biochemistry, Wol. 2. (Edited by FLORKIN M. & MASON H. S.) pp. 161-244. Academic Press, New York. COOPER W. K. (1962) The influence of time and ration composition on blood constituents and rumen mucosal glutamate synthesis in sheep. M.S. Thesis, West Virginia University, Morgantown, VA. DAS T. K. & WATERLOWJ. C. (1974) The rate of adaptation of urea cycle enzymes, aminotransferases and glutamic dehydrogenase to changes in dietary protein intake. Br. J. Nutr. 32, 353 373. DAVIDOVICHA., BARTLEYE. E., BECHTELR. M. & DAYTON A. D. (1977a) Ammonia toxicity in the cattle. III. Absorption of ammonia gas from the rumen and passage of urea and ammonia from the rumen to the duodenum. J. Anita. Sci. 46, 551-558. DAVIDOVICHA., BARTLEYE. E., MILLIKENG. A., DAYTON A. D., DEYOEC. W. & BECHTELR. M. (1977b) Ammonia toxicity in cattle. IV. Effects of unprocessed or extrusioncooked mixtures of grain and urea, biuret, or dicyanodiamide and liquid supplements on rumen and blood changes associated with toxicity. J. Anita. Sci. 45, 1397-1408. DIEM K. & LENTNER C. (1970) Scientific Tables, p. 573. Ciba-Geigy, Basle. EMMANUELB. & GILANPOURH. (1978) Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo. Comp. Biochem. Physiol. 61B, 28%289. HAMADA T., MAEDA S. & KAMEOKA K. (1976) Factors influencing growth of rumen, liver and other organs in kids weaned from milk replacers to solid foods. J. Dairy Sci. 59, 1110-1118. HARMEYERJ., KURELECB. & HILL H. (1968) Ueber Funktionen der Arginase, Urease und Ornithintranscarbamyl-
Urea cycle enzymes in sheep tissues ase der Pansenwand. 2. Mitteilung: Ornithintranscarbamylasefunktion. Zbl. Vet. Med. 15, 510-516. HOL~NIUS P. & JACOBSSON S-O. (1966) Ornithine-carbamyl transferase (OCT) activity in ruminants. Cornell Vet. 56, 187-195. HosmNo S., SARUMARUK. & MORIMATOK. (1966) Ammonia anabolism in ruminants. J. Dairy Sci. 49, 1523-1528. HOUPT T. R. & HOUPT K. A. (1968) Transfer of urea nitrogen across the rumen wall. Am. J. Physiol. 214, 1296-1303. HUNGATE E. R. (1968) The Rumen and its Microbes, pp. 281-330. Academic Press, New York. IDEY. (1969a) Enzymatic activities in ruminal mucosa. I. Surveys of carbamyl phosphate synthetase and ornithine transcarbamylase in goats. Jap. J. Vet. Sci. 31, 95-98. IDEY. (1969b) Enzymatic activities in ruminal mucosa. II. Presence and activities of L-glutamic dehydrogenase in goats. Jap. J. Vet. Sci. 31, 99 103. KRVAVICA S., KUERELECB. & MARTIN(~IC'T. (1964) Enzymatic investigations in the mucosa of the rumen. III. On the presence of carbamyl phosphate synthetase and ornithine transcarbamylase in the mucosa of the rumen. Vet Arh. Zagreb. 34, 94-100. KURELEC B., HARMEYERJ. & HILL H. (1968) Ueber Funktionen der Arginase, urease und Ornithintranscarbamylase der Pansenwand. 1. Mitteilung: Arginase-und Ureasefunktion. Zbl. Vet. Med. 15, 460-469. MARTIN~IC' T. & KRVAWCA S. (1964) Enzymatic investigations in the mucosa of the rumen. II. On the presence on arginase in the ruminal mucosa of cattle. Vet. Arh. Zaoreb. 34, 90-93. MATHISON G. W. & MILLIGAN L. P. (1971) Nitrogen metabolism in the sheep. Br. J. Nutr. 25, 353-366. McGILVERY P. W. (1970) Biochemistry. A Functional Approach, p. 361. Saunders, Philadelphia. MEHREZ A. Z., ORSKOVE. R. & MCDONALDJ. (1977) Rates of rumen fermentation in relation to ammonia concentration. Br. J. Nutr. 38, 437-443. NATALE P. J. & TREMBLAY G. C. (1974) Studies on the availability of intramitochondrial carbamylphosphate for utilization in extramitochondrial reactions in rat liver. Archs Biochem. Biophys. 162, 357 368. NOLAN J. V. (1975) Quantitative models of nitrogen metabolism in the sheep. In Digestion and Metabolism in the Ruminant (Edited by McDONALD I. W. & WARNER A. C. I.) pp. 416-431. The University of New England Publishing Unit, Armidale.
697
NOLAN J. V. & LENG R. A. (1972) Dynamic aspects of ammonia and urea metabolism in sheep. Br. J. Nutr. 27, 177-194. NOLAN J. V., NORTON B. W. & LENG R. A. (1976) Further studies of the dynamics of nitrogen metabolism in the sheep. Br. J. Nutr. 35, 127-147. SALEM H. A., DEVLIN T. J. & MARQUARDT R. R. (1973) Effects of urea on the activity of glutamate dehydrogenase, glutamine synthetase, carbamyl phosphate synthetase, and carbamyl phosphokinase in ruminant tissues. Can. J. Anita. Sci. 53, 503-511. SCHIMr,E R. T. (1962) Adaptive characteristics of urea cycle enzymes in the rat. J. biol. Chem. 237, 459~,68. SHIN V. E. & EFRO~ M. L. (1972) Urea cycle disorders. In The Metabolic Basis of Inherited Disease (Edited by STANBURY J. l., WYNGAARDEN J. B. & FREDRICKSON D. S.) pp. 370-392. McGraw-Hill, New York. SHIMBAYASHI K. & YONEMURAT. (1970) An aspect of urea cycle enzymes in goat. Agric. Biol. Chem. 34, 16031609. STEPrtEN J. M. L. (1968) Adaptive enzyme changes during protein depletion and refeeding. Br. J. Nutr. 22, 153 163. STEVEN D. H. & MARSHALLA. B. (1970) Organization of the rumen epithelium. In Physiology of Digestion and Metabolism in the Ruminant (Edited by PHXLLIPSONA. T.) pp. 80-100. Oriel Press, Newcastle-upon-Tyne. VAN DER HORST G. J. C. (1961) Keto acids and amines in the rumen liquid of cattle. Nature 191, 73 74. WHANGER P. D. & CHURCH D. C. (1970) Enzyme activities of ruminant digestive tract epithelial tissue. J. Dairy Sci. 30, 944948. WEEKST. E. C. (1972) Effects of pregnancy and lactation in sheep on the metabolism of propionate by the ruminal mucosa and some enzymic activities in the ruminal mucosa. J. Agric. Sci., Camb. 79, 409~,21. WEEKS T. E. C. (1974) The in vitro metabolism of propionate and glucose by the rumen epithelium. Comp. Biochem. Physiol. 49B, 393-406. YAVONENKOO. F., HZHYTS'KYIS. Z., IVANOVAL. H., BABIICrtUR A. M. & PYVOVAROVAA. O. (1972) Kinetic response of reductive amination of ketoglutaric acid in the cattle rumen wall. Dopov Akad. Nauk. ukr. RSR 34, 267-270. YAVONENKO O. F. (1969) Reductive amination of alphaketo-glutaric acid in the rumen wall of cattle. Ukr. Biokhim. Zh. 41,572-575.
0305-0491/80/0401-0693502.00/0
UREA CYCLE ENZYMES IN TISSUES (LIVER, RUMEN EPITHELIUM, HEART, KIDNEY, LUNG AND SPLEEN) OF SHEEP (OVIS ARIES) B. EMMANUEL Department of Animal Biology, School of Veterinary Medicine, University of Shiraz, Shiraz, Iran (Received 20 July 1979) Abstract--1. The enzymes of urea cycle were studied in the tissues (liver, rumen epithelium, heart,
kidney, lung and spleen) of sheep (Ovis aries). 2. The complete urea cycle was functional in significant quantities only in the liver. 3. Activities of urea cycle enzymes (carbamyl phosphate synthetase, ornithine transcarbamylase, arginine synthetase and arginase) were much higher in the liver than in other tissues; the enzymic pattern in other tissues tested was the same. 4. It is concluded that the rumen epithelium disposes very little ammonia through the urea cycle. Other mechanisms in addition to glutamate dehydrogenase and glutamine synthetase are expected to exist in this tissue to avoid ammonia toxicity. The rumen epithelium is exposed to very high concentrations of ammonia (5-55 mg NH3-N/100 ml rumen contents).
INTRODUCTION
The degradation of nitrogenous compounds by rumen micro-organisms leads to the production of high levels of ammonia in the rumen (Hungate, 1968; Church, 1971). The entry rates and concentrations of ammonia in the rumen of sheep fed conventional diets respectively were: 14-15g NH3-N/day (Nolan & Leng, 1972; Nolan et al., 1976) and 5-55mg NH3-N/100ml rumen contents (Hungate, 1968; Mathison & Milligan, 1971; Mehrez et al., 1977). Of the total ammonia entering the rumen pool about 40% is absorbed through the rumen wall, 46% is incorporated into microbial protein and 16% flows to the lower part of the digestive tract (Nolan, 1975). Interestingly enough, the levels of ammonia in the blood of ruminants and monogastrics are comparable {Diem & Lentner, 1970; Shin & Efron, 1972; Davidovich et al., 1977a). On the other hand, concentrations of ammonia in the rumen are 50-100 times higher than in portal blood (Bartley et al., 1976; Davidovich et al., 1977b). Therefore, highly efficient mechanisms of ammonia assimilation are expected to be present in the rumen epithelium to prevent ammonia toxicity in this tissue. The occurrence of the following enzymes, utilizing ammonia in the rumen epithelium has been reported: glutamate dehydrogenase (EC 1.4.1.3) (Cooper, 1962; Ide, 1969b; Yavonenko, 1969; Yavonenko et al., 1972), glutamine synthetase (EC 6.3.1.2) (Hoshino et al., 1966; Chalupa et al., 1970; Salem et al., 1973), and transaminases (glutamate-oxalacetate, glutamate-pyruvate, ornithine, tyrosine, tryptophan and phenylalanine (Chalupa et al., 1970; Whanger & Church, 1970; Weeks, 1972, 1974). Data on the presence of urea cycle enzymes in the rumen epithelium is not conclusive Arginase (EC 3.5.3.1) was found in the rumen epithelium of cattle (Martin~i6 & Krvavica, 1964; Kurelec et al., 1968; Whanger & Church, 1970), and goat (Kurelec et al., 1968), and ornithine transcarbamylase (EC 2.1.3.3) in the rumen wall of cattle (Hol-
tenius & Jacobsson, 1966; Harmeyer et al., 1968; Whanger & Church, 1970), and goat (Harmeyer et al., 1968; Ide, 1969a). Krvavica et al. (1964), and Ide (1969a) could not detect any activity of carbamyl phosphate synthetase (EC 2.7.2.5) in the rumen epithelium of cattle and goat while Salem et al. (1973) reported its presence in the rumen epithelium of calves. The activity of argininosuccinate synthetase (EC 6.3.4.5), and argininosuccinase (EC 4.3.2.1), and the complete function of the urea cycle have not been investigated in the rumen epithelium. No work has been carried out on the enzymes of urea cycle in the rumen epithelium of sheep. The present work was aimed to investigate the possibility of the operation of complete urea cycle (the incorporation of ammonia and COz into urea), and the activity of the five enzymes of this cycle in the rumen epithelium of sheep. Other tissues including liver, heart, kidney, lung and spleen were used for comparison. MATERIALS AND METHODS
L-[14C]guanido-arginine (55 mCi/mmol), L-[ ! 4C]ureidocitrulline (62mCi/mmol), and [t4C]sodium bicarbonate (59.9mCi/mmol) were purchased from Radiochemical Centre, Amersham, Bucks, U.K. L-citrulline-HC1,L-ornithine-HC1, L-arginine, N-acetyl glutamic acid, L-aspartic acid, 3-phosphoglyceric acid, glutathione, carbamyl phosphate and urease (type III) were the products of Sigma (London) Chemical Co. Tissue homogenate Tissues (rumen epithelium, liver, heart, kidney, lung and spleen) were obtained from Shiraz city abattoir immediately after slaughter, kept in ice and transferred within 45 rain to the laboratory. Rumen epithelium was stripped from the underlying tissues, washed with physiological saline a few times while stirring (to remove food particles and bacteria), and then blotted. A homogenate of all tissues was made of 1 part tissue and 9 parts of a solution of 0.025 M KH2PO4 (pH 7.8), using a Silverson homogenizer
693
694
B. EMMANt:EC
acid synthesis are located in the mitochondria and cytoplasm, respectively (McGilvery, 1970). However, carbamyl phosphate formed can freely pass the mitochondrial membrane (Natale et al., 1974). Both carbamyl phosphate synthetase and ornithine transcarbamylase are mitochondrial enzymes. The work of Ide (1969a) showing 14 times higher ornithine transcarbamylase specific activity in the mitochondrial fraction than whole rumen mucosa may indirectly indicate the presence of carbamyl phosphate synthetase in the rumen epithelial mitochondria. Regardless in what compartment carbamyl phosphate is formed, it can be further utilized for citrulline synthesis in the mitochondria. The activity of liver carbamyl phosphate synthetase was about 20 times higher than the rumen epithelium. Other tissues showed similar activities. Sheep liver activity was higher than that of cattle (5.79 units/g wet wt) (Krvavica et al., 1964), but lower than that of goat (487 557 units) (Brown & Cohen, 1960; Ide, 1969a). The activity of liver ornithine transcarbamylase was high (1877 units), about 55 82 times the values found in other tissues. The pattern in different tissues was similar to the data of Holtenius & Jacobsson (1966) performed on cattle tissues. Other workers (Krvavica et al., 1964; Whanger & Church, 1970) showed lower values (2(~110 units) in this species. On the other hand, activities reported for goat liver were 19,250-24,000 units (Cohen & Brown, 1960; Ide, 1969a). The activity in rat liver tested here was 5920 units/g wet wt, and the enzyme was completely dependent on ornithine and carbamyl phosphate. The activity in the rumen epithelium was respectively 2.4 and 4 times the values found in the goat (Ide, 1969a), and cattle (Krvavica et al., 1964; Whanger & Church, 1970). Activities of arginine synthetase which utilizes two consecutive reactions (argininosuccinate synthetase and argininosuccinase) were 50 and 14 units for the liver and rumen epithelium, respectively. The values of argininosuccinate synthetase found in the liver of goat and ox were 75 and 92 units, respectively (Brown & Cohen, 1960). RESULTS AND DISCUSSION Arginase activity in the rumen epithelium was 26.5 The results on enzyme activities are tabulated in units/g wet wt, The values reported for the rumen Table 1. Some activity (3 units/g wet wt) of carbamyl epithelium of cattle were 30-205 units (Martin6ic" & phosphate synthetase in the rumen epithelium was Krvavica, 1964; Whanger & Church, 1970). The value found. This finding agrees with the work of Salem et of 50.7 units found in the kidney agrees with that of al. (1973) performed on calves, but contrasts the cattle (Martin6ic' & Krvavica, 1964). The data on the results of Krvavica et al. (1964), and Ide (1969a) who spleen, heart, lung and kidney was of the same order. observed no activity in the rumen epithelium of the The liver had the highest activity which is in accord cattle and goats. In the present studies, whole rumen with the data on cattle liver (Martin6ic' & Krvavica, 1964). On the other hand, this value is much lower epithelial homogenate was used whereas, those authors utilized cell-free extracts. Previous work than that reported in earlier studies in goat liver (unpublished data) showed that 3-hydroxybutyrate (Brown & Cohen, 1960). From the literature data predehydrogenase (EC 1.1.1.30), a mitochondrial enzyme, sented here, it appears that there are species differis tightly bound to the cell particles in the rumen ences in ruminants with respect to enzyme activities epithelium, and is only partially released by liquid of urea cycle. The complete urea cycle which utilizes all five nitrogen and sonication treatments. Therefore, it is likely that in cell-free extract preparation, carbamyl enzymes was shown to be functional significantly only phosphate synthetase was not present. Carbamyl in the liver (700-850 times higher than other tissues). phosphate is needed not only for urea synthesis, but The activity (30 units/g wet wt) was lower than the also for nucleic acids formation. Thus, it is logical to activity of each enzyme separately. This is expected expect the presence of this enzyme in the rumen epith- since carbamyl phosphate synthetase, argininosuccielium which is metabolically an active tissue. The nate synthetase and argininosuccinase are rate limitcarbamyl phosphate synthetases for urea and nucleic ing steps in the urea cycle as shown in different
at a moderate speed. Whole homogenate was used for urea cycle enzyme assays. Arginase, arginine synthetase and complete urea cycle were assayed essentially the same as described previously (Emmanuel & Gilanpour, 1978) with very little modifications. The arginase (EC 3.5.3.1) assay consisted of 100 pmols of Tris buffer (pH 8.0), 50 #mols sodium glycinate (pH 9.5), 2.5 #mols L-arginine(pH 9.5), 0.5/aCi e-[14C]guanido-arginine, 0.5/~mols MnCIz, and 100 #1 tissue homogenate. The arginine synthetase (argininosuccinate synthetase plus argininosuccinase) assay contained 100pmols Trisbuffer (pH 8.0), 30 #mols MgSO4, 10 #tools L-aspartic acid, 5 pmols ATP (pH 7.0), 5 pmols L-citrulline-HCl, 0.5/xCi L-[14C]ureido-citrulline and 200 #1 tissue homogenate. The reaction mixture for the complete urea cycle assay was composed of 100 #tools Tris buffer (pH 8.0), 10 pmols ATP (pH 7.0), 10pmols L-ornithine-HC1, 10pmols Nacetyl glutamic acid, 30pmols MgSO4, 16 pmols reduced glutathione, 10#mols L-aspartic acid, l#mol MnC12, 5 pmols 3-phosphoric acid (to regenerate ATP), 10 pmols NaHI4CO3 (in 1 N NaOH to prevent escape of CO2). The reaction mixture was then made to pH 7.5 by adding 70 pl of 1N HCI, and 10pmols of NH4HCO3 and 100pl of tissue homogenate were added. The subsequent procedures up to the counting of the radioactivity were the same as described previously (Emmanuel & Gilanpour, 1978). The ornithine transcarbamylase (EC 2.1.3.3) assay contained 300,umols Tris-buffer (pH 8.0), 10pmols L-ornithine HCI, 10/~mols carbamyl phosphate, 2 mg urease and 100 pl of tissue homogenate in a total volume of 3 ml. The reaction mixture was incubated in a shaking water bath at 37°C for 30min. At the end of the incubation time, the reaction was terminated by adding 2 rnl of 15% metaphosphoric acid. The determination of the product (citrulline) formed was made as described by Archibald (1944). The carbamyl phosphate synthetase (EC 2.7.2.5) assay consisted of 300 #mols Tris buffer (pH 8.0), 25/~mols ATP (pH 7.0), 20 pmols L-ornithine-HCl, 25/tmols N-acetyl glutamic acid (pH 7.0), 16 pmols reduced glutathione (pH 7.0), 30 pmols MgSO4, 12.5,umols 3-phosphoglyceric acid (pH 7.0), 50 pmols NH4HCO3 and 250 pl of tissue homogenate in a total volume of 3ml. The citrulline formed was measured as described above. Since all tissues tested had an active ornithine transcarbamylase, the addition of this enzyme commercial was unneccessary.
Urea cycle enzymes in sheep tissues
695
Table 1. Activities (units*/g wet wt) of urea cycle enzymes in the sheep (Ovis aries) tissues Enzyme Tissue Liver
Mean + SD
Carbamyl phosphate synthetase 60.4 75.4 47.2 --61.0 14.1
Spleen
7.10 6.91 7.62 --
Mean + SD Lung
Mean _ SD Heart
Mean ___SD Kidney
Mean + SD Rumen epithelium
Mean + SD
Ornithine transcarbamylase 1524 1646 1783 1890 2540 1877 396
Arginine** synthetase 56.7 52.2 50.0 42.4 48.3 49.9 5.25
Arginase 648 613 545 310 470 517 134
Completer urea cycle 27.4 14.6 39.8 22.7 44.3 29.8 12.1
32.5 24.4 32.6 34.4 47.5 34.3 8.34
89.3 13.2 46.7 58.4 81.9 57.9 30.4
0.048 0.039 0.043
7.21 0.36
30.7 30.5 32.4 37.0 24.8 31.1 4.38
6.90 8.38 4.57 --6.62 1.91
30.5 32.4 48.6 25.7 22.9 32.0 10.03
36.0 23.7 28.7 31.2 34.8 30.9 4.95
54.7 25.4 49.2 73.0 75.8 55.6 20.4
0.030 0.048 0.037
5.70 6.10 4.57 --5.46 0.78
27.4 36.2 32.4 23.8 30.4 30.0 4.71
37.0 31.7 20.6 39.4 40.2 33.8 8.08
50.6 26.7 48.5 57.0 78.0 52.2 18.4
0.031 0.037 0.033
5.81 6.10 6.86 --6.25 0.57
24.4 22.3 24.8 17.1 26.7 23.1 3.68
35.7 26.6 36.0 35.6 50.1 36.8 8.42
27.2 49.7 45.6 52.9 78.2 50.7 18.3
0.031 0.045 0.028 ---0.035 0.009
3.00 3.81 2.29 --3.03 0.79
33.3 36.2 28.6 34.3 36.2 33.7 3.12
16.9 11.1 14.5 12.9 13.1 13.7 2.16
18.2 30.6 28.9 19.5 35.1 26.5 7.23
0.038 0.032 0.035 --0.035 0.003
-0.043 0.005
0.038 0.009
-0.034 0.003
* Unit of enzyme activity is expressed as 1 ,umol product formed/hr at 37°C. ** Arginine synthetase consists of the activities of argininosuccinate synthetase plus argininosuccinase. 1"Complete urea cycle utilizes all the reacions leading from ammonia and CO2 to the formation of urea. species (Brown & Cohen, 1960). The complete urea cycle developed (Emmanuel & Gilanpour, 1978) is very sensitive. The reaction was completely dependent on ATP, N-acetyl glutamic acid, magnesium and tissue homogenate. The activity was reduced by 70, 84 and 94~o in the absence of ornithine, N H 4 H C O 3 , and aspartic acid, respectively. Maximal activity was shown at p H 7.5. It is i m p o r t a n t to note that the results of arginine synthetase, arginase a n d complete urea cycle assays presented here may have beeVn underestimated. As described in the Materials a n d M e t h o d s section (Emmanuel & Gilanpour, 1978), in these enzyme assays, the product formed ([14C]urea) was measured enzymatically, using commercial urease. The presence of urease c.B.P. 65/4B--H
of bacterial origin which is hypothesized to be diffused from the r u m e n into the epithelial cells has been reported (Abdel R a h m a n & Decker, 1966; H o u p t & Houpt, 1968). Therefore, if urease activity was present in the r u m e n epithelial homogenate of the present work, [14C]urea could be hydrolysed to 14CO2 and a m m o n i a during the incubation period, and the former could have been released after acidification, thus resulting in lower values for those enzymes. Nevertheless, the activities of carbamyl p h o s p h a t e synthetase and ornithine transcarbamylase (which are not affected by the presence of urease) in the r u m e n epithelium were 20 a n d 55 times lower than respective values in the liver. The above a r g u m e n t points to the insignificant function of the complete urea cycle in
696
B. EMMANUEL
this tissue. Chalupa et al. (1970) suggested the presence of trace amounts of five urea cycle enzymes in the rumen wall of the sheep. Under certain dietary regimens, the synthesis of urea cycle enzymes in the rumen epithelium may be induced, therefore, contributing more to ammonia assimilation in this tissue. Sheep fed on soy protein diet had higher activities of transaminases and glutamate dehydrogenase in the rumen epithelium than animals fed on a urea diet (Chalupa et al., 1970). Similarly, rumen epithelium glutamine synthetase activities were higher in calves receiving 50~o u r e a - N diet than 0, or 100°~o urea-N diets (Salem et al., 1973). Adaptation characteristics of NH3-assimilating enzymes on dietary protein have been reported in goat liver (Shimayashi & Yonemura, 1970), and rat liver (Schimke, 1962; Stephen, 1968; Das & Waterlow, 1974). Considering the subject from a different angle, the occurrence of some of the urea cycle enzymes may not necessarily point on the operation of the cycle in the tissue under question. As mentioned above, carbamyl phosphate synthetase found in the rumen epithelium may be functional more in nucleic acids than urea synthesis. Hameyer et al. (1968) suggested that urease, arginase and ornithine transcarbamylase operate cooperatively in the rumen epithelium in recirculation of nitrogen compounds between the rumen contents and the blood. Similar to urea cycle enzymes, the activities of transaminases and glutamate dehydrogenase were also lower in the rumen epithelium than in the liver (Whanger & Church, 1970; Salem et al., 1973). Considering this, and the facts that the liver is twice as heavy as the rumen epithelium (Hamada et al., 1976), and ammonia levels in the rumen are markedly higher than in the blood (Bartley et al., 1976), it appears that the above mentioned NH3-assimilating mechanisms are not efficient enough to explain ammonia detoxication in the rumen epithelium. Rumen epithelium is metabolically an active tissue; it is composed of cells containing numerous mitochondria, rough endoplasmic reticulum, Golgi apparatus and large nuclei with prominent nucleoli (Steven & Marshall, 1970). Perhaps the rumen has developed sophisticated mechanisms for ammonia disposal in order to avoid ammonia toxicity. Other mechanisms of NH3-fixation which have not been referred to in the text are asparagine synthetase and reductive amination of keto-acids (such as pyruvate to alanine, and glyoxylate to glycine which occur in bacteria (Cohen & Brown, 1960)). Branched-chain amino acids, phenylalanine and tryptophan in rumen micro-organisms are synthesized through carboxylation of respective keto-acids and then amination (Allison, 1970). Van der Horst (1961) has reported the presence of keto-acids including 2-ketoisovalerate, 2-ketoisocaproate, 2-keto-3-methylvalerate, hydroxypyruvate and glyoxylate in rumen contents. These keto acids may be transaminated or reductively aminated in the rumen epithelium. Further work is required to verify other mechanisms of NH3-assimilation in the rumen epithelium. It is concluded that little amounts of all five urea cycle enzymes were found in the rumen epithelium, however, the function of complete urea cycle in the rumen epithelium is insignificant as compared to the
liver. Other tissues tested showed similar characteristics of urea cycle enzymes as the rumen epithelium. Acknowledgements--The author wishes to thank Mr T. Vaseghee for his excellent technical assistance. This work was supported by a grant from University of Shiraz Research Council.
R EFERENCES
ABDEL RAHMANS. (~ DECKER P. (1966) Comparative study of urease in the rumen wall and rumen contents. Nature, Lond. 209, 618-619. ALLISON M. J. (1970) Nitrogen metabolism of ruminal micro-organisms. In Physiology of Digestion and Metabolism in the Ruminant (Edited by PHILLIPSON A. T.) pp. 456-473. Oriel Press, Newcastle-upon-Tyne. ARCHIBALD R. M. (1944) Determination of citrulline and allantoin and demonstration of citrulline in blood plasma. J. biol. Chem. 156, 121-142. BARTLEYE. E., DAVIDOVICH A. D., BARR G. W., GRIEFEL G. W., DAYTONA. D., DEYOEC. W. & BECHTLE R. M. (1976) Ammonia toxicity in cattle. I. Rumen and blood changes associated with toxicity and treatment methods. J. Anita. Sci. 43, 835-841. BROWN JR G. W. & COHEN P. P. (1960) Comparative biochemistry of urea synthesis. 3. Activities of urea-cycle enzymes in various higher and lower vertebrates. BiDchem. J. 75, 82-91. CHALUPA W., CLARK J., OPL1GER P. & LAVKER R. (1970) Ammonia metabolism in rumen bacteria and mucosa from sheep fed soy protein or urea. d. Nutr. 100, 161 169. CHURCH D. C. (1971) Digestive Physiology and Nutrition of Ruminants, Vol. 1. pp. 199 223. O.S.U. Book Stores, Corvallis. COHEN P. P. & BROWNJR G. W. (1960) Ammonia metabolism and urea biosynthesis. In Comparative Biochemistry, Wol. 2. (Edited by FLORKIN M. & MASON H. S.) pp. 161-244. Academic Press, New York. COOPER W. K. (1962) The influence of time and ration composition on blood constituents and rumen mucosal glutamate synthesis in sheep. M.S. Thesis, West Virginia University, Morgantown, VA. DAS T. K. & WATERLOWJ. C. (1974) The rate of adaptation of urea cycle enzymes, aminotransferases and glutamic dehydrogenase to changes in dietary protein intake. Br. J. Nutr. 32, 353 373. DAVIDOVICHA., BARTLEYE. E., BECHTELR. M. & DAYTON A. D. (1977a) Ammonia toxicity in the cattle. III. Absorption of ammonia gas from the rumen and passage of urea and ammonia from the rumen to the duodenum. J. Anita. Sci. 46, 551-558. DAVIDOVICHA., BARTLEYE. E., MILLIKENG. A., DAYTON A. D., DEYOEC. W. & BECHTELR. M. (1977b) Ammonia toxicity in cattle. IV. Effects of unprocessed or extrusioncooked mixtures of grain and urea, biuret, or dicyanodiamide and liquid supplements on rumen and blood changes associated with toxicity. J. Anita. Sci. 45, 1397-1408. DIEM K. & LENTNER C. (1970) Scientific Tables, p. 573. Ciba-Geigy, Basle. EMMANUELB. & GILANPOURH. (1978) Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo. Comp. Biochem. Physiol. 61B, 28%289. HAMADA T., MAEDA S. & KAMEOKA K. (1976) Factors influencing growth of rumen, liver and other organs in kids weaned from milk replacers to solid foods. J. Dairy Sci. 59, 1110-1118. HARMEYERJ., KURELECB. & HILL H. (1968) Ueber Funktionen der Arginase, Urease und Ornithintranscarbamyl-
Urea cycle enzymes in sheep tissues ase der Pansenwand. 2. Mitteilung: Ornithintranscarbamylasefunktion. Zbl. Vet. Med. 15, 510-516. HOL~NIUS P. & JACOBSSON S-O. (1966) Ornithine-carbamyl transferase (OCT) activity in ruminants. Cornell Vet. 56, 187-195. HosmNo S., SARUMARUK. & MORIMATOK. (1966) Ammonia anabolism in ruminants. J. Dairy Sci. 49, 1523-1528. HOUPT T. R. & HOUPT K. A. (1968) Transfer of urea nitrogen across the rumen wall. Am. J. Physiol. 214, 1296-1303. HUNGATE E. R. (1968) The Rumen and its Microbes, pp. 281-330. Academic Press, New York. IDEY. (1969a) Enzymatic activities in ruminal mucosa. I. Surveys of carbamyl phosphate synthetase and ornithine transcarbamylase in goats. Jap. J. Vet. Sci. 31, 95-98. IDEY. (1969b) Enzymatic activities in ruminal mucosa. II. Presence and activities of L-glutamic dehydrogenase in goats. Jap. J. Vet. Sci. 31, 99 103. KRVAVICA S., KUERELECB. & MARTIN(~IC'T. (1964) Enzymatic investigations in the mucosa of the rumen. III. On the presence of carbamyl phosphate synthetase and ornithine transcarbamylase in the mucosa of the rumen. Vet Arh. Zagreb. 34, 94-100. KURELEC B., HARMEYERJ. & HILL H. (1968) Ueber Funktionen der Arginase, urease und Ornithintranscarbamylase der Pansenwand. 1. Mitteilung: Arginase-und Ureasefunktion. Zbl. Vet. Med. 15, 460-469. MARTIN~IC' T. & KRVAWCA S. (1964) Enzymatic investigations in the mucosa of the rumen. II. On the presence on arginase in the ruminal mucosa of cattle. Vet. Arh. Zaoreb. 34, 90-93. MATHISON G. W. & MILLIGAN L. P. (1971) Nitrogen metabolism in the sheep. Br. J. Nutr. 25, 353-366. McGILVERY P. W. (1970) Biochemistry. A Functional Approach, p. 361. Saunders, Philadelphia. MEHREZ A. Z., ORSKOVE. R. & MCDONALDJ. (1977) Rates of rumen fermentation in relation to ammonia concentration. Br. J. Nutr. 38, 437-443. NATALE P. J. & TREMBLAY G. C. (1974) Studies on the availability of intramitochondrial carbamylphosphate for utilization in extramitochondrial reactions in rat liver. Archs Biochem. Biophys. 162, 357 368. NOLAN J. V. (1975) Quantitative models of nitrogen metabolism in the sheep. In Digestion and Metabolism in the Ruminant (Edited by McDONALD I. W. & WARNER A. C. I.) pp. 416-431. The University of New England Publishing Unit, Armidale.
697
NOLAN J. V. & LENG R. A. (1972) Dynamic aspects of ammonia and urea metabolism in sheep. Br. J. Nutr. 27, 177-194. NOLAN J. V., NORTON B. W. & LENG R. A. (1976) Further studies of the dynamics of nitrogen metabolism in the sheep. Br. J. Nutr. 35, 127-147. SALEM H. A., DEVLIN T. J. & MARQUARDT R. R. (1973) Effects of urea on the activity of glutamate dehydrogenase, glutamine synthetase, carbamyl phosphate synthetase, and carbamyl phosphokinase in ruminant tissues. Can. J. Anita. Sci. 53, 503-511. SCHIMr,E R. T. (1962) Adaptive characteristics of urea cycle enzymes in the rat. J. biol. Chem. 237, 459~,68. SHIN V. E. & EFRO~ M. L. (1972) Urea cycle disorders. In The Metabolic Basis of Inherited Disease (Edited by STANBURY J. l., WYNGAARDEN J. B. & FREDRICKSON D. S.) pp. 370-392. McGraw-Hill, New York. SHIMBAYASHI K. & YONEMURAT. (1970) An aspect of urea cycle enzymes in goat. Agric. Biol. Chem. 34, 16031609. STEPrtEN J. M. L. (1968) Adaptive enzyme changes during protein depletion and refeeding. Br. J. Nutr. 22, 153 163. STEVEN D. H. & MARSHALLA. B. (1970) Organization of the rumen epithelium. In Physiology of Digestion and Metabolism in the Ruminant (Edited by PHXLLIPSONA. T.) pp. 80-100. Oriel Press, Newcastle-upon-Tyne. VAN DER HORST G. J. C. (1961) Keto acids and amines in the rumen liquid of cattle. Nature 191, 73 74. WHANGER P. D. & CHURCH D. C. (1970) Enzyme activities of ruminant digestive tract epithelial tissue. J. Dairy Sci. 30, 944948. WEEKST. E. C. (1972) Effects of pregnancy and lactation in sheep on the metabolism of propionate by the ruminal mucosa and some enzymic activities in the ruminal mucosa. J. Agric. Sci., Camb. 79, 409~,21. WEEKS T. E. C. (1974) The in vitro metabolism of propionate and glucose by the rumen epithelium. Comp. Biochem. Physiol. 49B, 393-406. YAVONENKOO. F., HZHYTS'KYIS. Z., IVANOVAL. H., BABIICrtUR A. M. & PYVOVAROVAA. O. (1972) Kinetic response of reductive amination of ketoglutaric acid in the cattle rumen wall. Dopov Akad. Nauk. ukr. RSR 34, 267-270. YAVONENKO O. F. (1969) Reductive amination of alphaketo-glutaric acid in the rumen wall of cattle. Ukr. Biokhim. Zh. 41,572-575.