pp. 385-389, 1992
~TRIBUTION IN TISSUE', ANIMALS V[AHMOUD AMINLARI*
and
)MESTIC
TALEB VASI
ry, School of Veterinary Medicine, Shiraz
iraz, 71365 Iran
Received 6 March 1992; accepted 8 April 1 ofal A~tract--1. A new colorimetric method was usedI for determination determi domestic animals. 2. In all species studied liver was the richest source of arginase. 3. Significant differences were observed in the specific activity activi of argin~ 4. In all species, besides liver, kidney and brain also con contained signi 5. In the dog, in addition to the three organs mentioned aabove, lung, showed some arginase activity. in 6. In sheep and cattle significant arginase activity was observed c observed between epithelial and muscular layers of different parts 1z of dige 7. These results are discussed in terms of the possible role rol of argina
INTRODUCTION
rginase (L-arginine amidinohydrolase, EC 3.5.3.1) is terminal enzyme of the urea cycle which catalyzes thee hydrolysis of L-arginine into urea and L-ornithine (Greenber ;reenberg, 1960). An extraordinarily extensive literature ure is available on arginase and many aspects of this is enzyme from different sources have been studied, Besides :sides its involvement in ammonia detoxification whir hich occurs through the urea cycle, the role of ar ginase in other processes such as cell toxicity due to decreasin :creasing arginine in macrophage culture (Currie, 1971 ~78), inhibitory effects on lymphoma cells in vitro (Reyero and Doner, 1975; RLuggs u and Russell, 1980), and its elevated level in growmg wing tissues and tumors (Taylor and Stewart, 1981, and references therein) has been reported. F u r t h e rrmore, m o r e arginase plays a key role in the biosynthesis; of polyamines in brain ~es (Vanella et al., 1979). and other extrahepatic tissues This enzyme is widely distributed xibuted in nature, from bacteria to man (Greenberg,g, 1960; Cornelius, 1980; De Ruitter and Kollofel, 1982; 82; Borkovich, 1987, and references therein). Atthou gh the distribution and properties of arginase from many tissues of human (Dahlig et al., 1975; Reyeero and Doner, 1975; ~75; Borcic and Straus, Van Elsen and Leroy, 1975; 1976; Porta et al., 1976; Snelman et al., 1979; ~80; Spector et al., 1982, Baranczyk-huzma et al., 1980; 1983; Konarsh et al., 1985; Zamecka cka and ~amecka and Porembska, 1988) and laboratory animals mls (Chen and Broome, 1980; Brusdeilins et al., 1985 )85; Spolaris and Bond, 1988, 1989) have been extensiv, ensively studied, little is ~tribution of this enzyme known of the pattern of distributio~ in the tissues of domestic animals. stigation was to examine The purpose of this investi gtribution of arginase in and compare the tissue distributio~ Jlts of these studies will domestic animals. The results help to assess the role of ar ginase in different tissues [ n ee
*To whom correspondence should be a~
ent tissues of some n different species. arginase. nd skeletal muscle '
o differences were all species studied. tissues of animals.
of animals. ani We , shown that the activity of rhodanese rh rnoaanesc (~.t~ 2.5.1.1) ana md beta-mercaptopyru~t C vate sulfurtransferase (EC 2.8.1.2!), two enzymes invob involved in cyanide detoxification, is higher in rumen epith( fithelium than in liver (Aminla Aminlari et al., 1989; Amin Aminlari and Gilanpour, 1991). In the l present study we tl tried to find out whether suc such a pattern of distril distribution exists for arginase. This " paper also repor 3orts on the application of a new t method for argin~ inase which has been developed )ed il in this laboratory. This simple colorimetric method is based on the deten determination of the remaining arginine, al after its hydr( hydrolysis, by reaction with p-nitrophenyl p-nitt glyoxal (PNPG) (Aminlari, 1992). This method met allows rapid determination of arginase in a hlarge number of samples. METHODS MATERIALS AND METH
All chemicals were of analytical grade and were supplied by commercial sources, p-Nitrophenyl gl glyoxal (PNPG) was synthesized as described previously (Aminlari, 1992). Organs of sheep (Ovis aries), cattle (Bos 0 taurus), camel (Camelus dromedaries), were obtained from slaughtered animals. Post-mortem horse (Equus caballus ), donkey familiaris) samples were (Equus asinus), and dog (Canis familk obtained from Veterinary School hospifital. These samples were prepared by autopsy of animals, 0-2 0-~ hr after death. All samples, kept on ice, were transferred within w 45 min to the laboratory; tissues were separated, stripped stril from fat and extraneous materials, washed a few time,, with physiological few times saline and then blotted. Tissue extracts extracts were prepared by freezing 1 g of the sample in liquid nitrogen, nitre homogenizing pending the homogenate with a hand-homogenizer, and suspendi in 4 ml of 0.025 M sodium phosphate buffer, I pH 7.2. The min at 4000 g in an suspensions were centrifuged for 15 m ge. The supernatants MSE high-speed refrigerated centrifuge were used as the source of enzyme. The muscular and epithelial layers of different partsLsof stomach ston and small and large intestine were separated and treated tr, as described _1_ . . . . A __: . . . . . . . . . . . . . . . . .I L . . .i. t by the PNPG method lari, 1992) with some
MAHMOUDAM1NLARI and TALEB VASE(
at pH 9.5 was added e was kept at 37°C for ieved by adding 0.2 ml ntaining 0.1 M sodium m ascorbate, pH9.0. volume of the reaction t distilled water and the gainst a blank containg a Unicam Model SP tandard curve a molar cm was obtained,
Determination o f arginase activity To a 0.2 ml solution of 20 mM arginine at pH 9.5 was :lded 20 pl 10% MnCI 2 and the mixture was warmed in a ater bath at 37°C for 5 min. Incubation was started by :lding 10-/d tissue extract and holding the mixture at 37°C )r 20 min. These solutions were treated for color developtent as described above. The concentration of the remainLg arginine was determined using the molar extinction )efficient (see above). For each sample, a control solution as routinely included which contained all solutions except tat no arginine was present. This control was included in rder to eliminate any interference caused by the reaction of NPG with constituents in the serum (such as arginine sidues of proteins). The absorbance due to this control was tbtracted from the absorbance of respective samples before dculating the pmol arginine remaining. One unit of •ginase activity was defined as #mol arginine hydrolyses ~r min at 37°C and pH9.5 and was calculated from following equation: Unit activity =/~mol arginine/min = (AA4s0/2.6 x 103) x 104 × 1/T. InL the above equation AA4s0 is the difference of the ab)rbance at 480 nm of a solution of arginine in the absence sorbance an(ld presence of arginase, 2.6 x 103 is the arginine-equivalent molar olar extinction coefficient, 104 is the factor for converting
mol/1 arginine to and T is the inc
in a 10 ml reaction mixture, usually 20 min).
C. Protein deten Protein was d 0953).
the method o f Lowry et al.
,TS A r g i n i n e rea pyrophosphate p H 9.0 a n d 37
4PG in 0.1 mol/l s o d i u m s o d i u m a s c o r b a t e , at ce a c o l o r e d c o m p o u n d which absorbe at 480 n m with a m o l a r eextin x t i n c t i o n coef x 103 M / c m . T h e maxim m absorbal t i n e d after 3 0 m i n . T h e m uu m rreact eaction descr was used to d e t e r m i n e m a s e activil argin aracts. A f t e r a d d i t i o n o f a ssan ample conta e to a p r e - m i x e d arginine solut solution and i r 20 min, the r e m a i n i n g arginine was react with P N P G . T h e diffel difference in tl: ,~ o f a n arginine s o l u t i o n in the absen( ~ence o f arginase was ccorre o r r e l a t e d to t] ' arginase in the sample. T h e specific ginase in different tissues Th d o m e s t i c an vn in Tables 1 a n d 2. In o f de species ;r was f o u n d to be the all the tl fiche: source richest In the dog, the levels o f argin mase in kidney, brain, spleen, hhee a r t a t r i u m , h e a r t ventr ventricle, a n d skeletal muscle are silgnificantly higher t h a n in o t h e r tissues, except the liver ( P < 0.01) (Tab'~ 1). In all species studied,, kkid1 (Table i d n e y a n d b r a i n are two o( r g a n s w h i c h c o n t a i n significm aificant activity o f this enzyi~me as c o m p a r e d to o t h e r o r g aans r listed. In sheep a n d cattle, a significant level o f arginase is also 9resent in the r u m e n (Table 2). N o significant differprese ence was o b s e r v e d in the arginase c o n t e n t b e t w e e n the m rr u s c u l a r a n d epithelial layers rers o f different p a r t s o f
Table 1.. Mean (_+SD) of the specific activity of arginase in the crude extracts from different tissues of dog, horse and donkey "I~^L.I^
1
Tissues
ltm~--
/
t
or'~\
_$'.l.
. . . . .
:~-
Dog N=5
~--,1..1
....
e-.
Units/mg protein Horse N=2
Donkey N=4 0.37 (0.04)*t 0.06 (0.010):~ 0.02 (0.008) 0.02 (0.007) O.02 (O.O02) 0.06 (0.020):~ O.O2 (O.OIO) O.O2 (O.OO9)
Liver 0.41 (0.07)*'[" 0.49 (0.04)*t Kidney 0.08 (0.01):~ 0.06 (0.002):~ Heart ventricle 0.08 (0.040)J; 0.03 (0.003) Lung 0.05 (0.020)~ 0.03 (0.003) Spleen 0.06 (0.010);~ 0.01 (0.002) Brain 0.06 (0.020):~ 0.06 (0.010)~ Heart atrium 0.09 (0.040)* 0.03 (0.006) Skeletal muscle 0.08 (0.030)~: 0.04 (0.007) Cardiac region of stomach 0.02 (0.004) Pyloric region of stomach 0.02 (0.010) Glandular part of stomach 0.02 (0.008) 0.01 (0.004) Preventricular part of stomach -0.01 (0.006) 0.02 (0.010) Duodenum 0.02 (0.010) 0.01 (0.001) 0.02 (0.015) 0.01 (0.006) 0.02 (0.006) 0.01 (0.007) Ileum 0.01 (0.007) 0.01 (0.003) 0.01 (0.004) Jejunum Colon 0.02 (0.010) 0.01 (0.002) 0.02 (0.010) Cecum 0.02 (0.005) 0.01 (0.008) 0.02 (0.014) Rectum 0.02 (0,010) ND ND N = Number mber of animals studied. ND = Not determined. *Value of this tissue is significantly different from all other tissues (P < 0.01). tThis value alue in the horse is significantly different from that of donkey (P < 0.01). :[:In all species, val but not signifi dpt (P < 0.01). -
-
-
-
-
Jes)
Arginase distribution + SD) of the specificactivityof arginasc in th different tissues of sheep, cattle and camel Units/mg protein Cattle Sheep
Omasum Reticulum Rumen Abomasal fundus Abomasal pylore Duodenum lleum Jejunum Colon Rectum
N=5
N=5
0.42 (0.03)*?
0.4o (0.o4)*?
0.08 (0.006)~:
0.09 (0,009):~
0.05 (0.010) 0.02 (o.001) 0.02 (0.001)
0.03 (0.009) 0.o4 (0.008) 0.03 (0.0O7)
0.08 (0.02):~ 0.04 (0.009) 0.02 (0.006) O.O1 (0.006)
0.08 0.03 0.02 0.02
0.03 (0.010)
0.o4 (0.009)
0.05 (O.OlO)?:l: 0.02 (0.006)
0.06 (0.008)Ui 0.03 (0.008)
0.03 (0.004)
0.02 (0.006)
0.02 0.03 0.01 0.01 0.01
0.01 0.02 0.01 0.01 0.01
(0.008) (0.010) (0.003) (0.005) (0.006)
(0.010):~ (0.004) (0.008) (0.008)
(0.009) (0.005) (0,006) (0.007) (0.006)
N = Number of animals studied. *Value of this tissue is significantly different from all ," other tisst ?These values in sheep and cattle are significantlydiff different from th :[:In all species, values of these tissues are significantly significanl different t not significantly different from values beating the th( same letter
igestive systems of all animals studied (data not town). No intra- or inter-species differences were Observe(1 bserved in arginase activity of any other tissues of animals listed in Tables 1 and 2.
DISCUSSION
In this paper, we describe the application of a new me tethod for the determination of arginase in different tissues ssues of domestic animals. This method is simpler and ad more rapid than other available methods for argmase, and uses fewer chemicals. I this method the s. In micals. sample containing arginase is added to a premixed incubation mixture and color or formation is achieved by the addition of a single reagent, i.e. PNPG. This compound performs two fun( notions. It reacts with the remaining arginine in the reaction mixture and, in effect, stops the arginase activit ctivity. Furthermore, the reaction product of arginine Le with PNPG is colored and can be used to calculate late arginase activity as described in the Methods section. These features greatly simplify the method ~d for determination of arginase and allow the screenin ning of a large number of samples in a short period of tim, time. The PNPG method has enough sensitivity to be.~used to compare tissues with little arginase activity. The data presented in thi:s communication indicate that liver is the richest source',e of o f arginase. In liver, the level of arginase is at least one order of magnitude greater than most other tissu( rues. The role of arginase in the metabolic life of cells ~ells has generally been considered in terms of its function unction in the urea cycle, In mammals, the liver is the ot organ in which a full urea cycle is functional (Greenber erg, 1960). However, the metabolic function of arginase m extrahepatic tissues use in is still unclear. The presence ce of arginase in tissues athwav~ fnr di~na~ino af of organisms using other pathways nitrogen has been explained as a deletion or repression of other enz,~
from
,? )~ ) ) ) )~ ) ) ) )t ) ) ) ) ) ) ) 0.01). s, but
O.Ol).
cycle (Brown I IJIVV'Vll O a. l t t . l k . . U t l ~ t l , I y960; l/V, •Vl Mora et al., 1965). Our, data show that besides liver, some s( other tissues of domestic animals show significant activity of argin mase. In all animals, kidney and brain also show considerable arginase activity. The presence of argin mase in extrahepatic tissues mitght indicate that these tissues use arginase for purposes puq other than urea synthesis. It has been reported )orted that for chickens (Emmanuel and Gilanpour, 1978), certain insects tHou House, 1965) and cells in tissue culture cult~ (Nesheim and Garli Garlich, 1963), arginine is an essenti essential nutrient. Dietary arginine serves as the sole source sol of ornithine which, in turn, can be metabolized to either proline or glutamate. Arginase can therefor~ herefore function physiologically in the metabolism of arlginine to proline and/or glutamate. Ornithine is also alst important as a precursor for polyamine biosynthesi ,nthesis which is necessary for cell division and differenti~ fferentiation. Such functions might be significant in the brain bra (Vanella et aL, 1979). A high arginase level in br brains of domestic animals might be due to its role in this regard. Our data show the presence of ar!ginase in different parts of the digestive system of sheep, ~, cattle and levels No significant camels, albeit at very low levels. difference is observed in the level of arginase between the epithelial and the muscular latyers of different parts of the digestive system of r~ ruminants. As we have reported earlier, rumen epithelium shows considerable activity of cyanide-i :anide-metabolizing enzymes, rhodanese and beta-mercapt tptopyruvate sulfurtransferase (Aminlari et al., 1989, Aminlari and Gilanpour, 1991). Although the rm rumen of sheep and cattle contains some arginase (Table (Tab 2), it probably does not play a significant role in aammonia detoxification as compared with liver, In h ruminants, the degradation of nitrogenous comp, ?pounds by rumen microorganisms leads to the produc )roduction of high levels c~f ~ r n m c m i ~ in t h p rnm@n [tr~hnro (Church, 1971). Highly amonia assimilation are n the rumen to prevent
MAHMOUD AMINLARI and TALEBVASEG
o u r results a n d the gators (Emmanuel, anger and Church, ruminants, arginase are n o t the main dfication in the rum o n i a in the r u m e n dehydrogenase (EC 1969), glutamine pula et al., 1970; saminases ( C h a p u l a anger a n d Church, )70), m i g h t be involved in this mechanism. As liver the richest source o f arginase, it would be expected lat the availability o f a simple m e t h o d for assaying lis enzyme, such as the one described in this paper, :nders arginase a suitable candidate for liver ruction tests in domestic animals. This idea is u n d e r arrent investigation in this laboratory• cknowledgements--This research was financially sup~rted by Grant No. 68-VE-523-281 from the Research ouncil of Shiraz University. Part of the data presented in ds paper was taken from theses by P. Najmodini, • Khalesi and M. Abdolhouseinzadeh.
REFERENCES minlari M. (1992) A novel colorimetric method for assaying arginase. Clin. Biochem. (in press). Aminl minlari M., Gilanpour H., Vaseghi T. and Taghavianpour H. (1989) Comparative studies on the distribution of rhodanese and beta-mercaptopyruvate sulfurtransferase in different organs of sheep and cattle. Comp. Biochem. Physiol• 92C, 259-262. Aminl minlari M. and Gilanpour H. (1991) Comparative studies on the distribution of rhodanese in different tissues of domestic animals. Comp. Biochem. Physiol• 99B, 673-677. Barancz ~ aranczyk-huzma A., Skarzypech-osiecha I., Zalejska M. and Porembska Z. (1980) Purification and some properproperties of human heart arginase. Lse. Acta Biochem. Pol. 27, 181-187. Borcic O. and Straus B. (1976) 76) Separation of arginase isoenzymes from human tissues ues by agar gel electrophoresis. J. clin. Chem. clin. Biochem. :hem. 14, 533-535. Brusdeilins M., Kuhner R. and tnd Schumacher K. (1985) Purification, atfinity to anti-human arginase immunoglobulin-Sepharose 4B B and subunit molecular weights of mammalian arginases. rases. Biochim. biophys. Acta 840, 79-90• Borkovich K. A. and Weiss R. L. (1987) Relationship between two major immunoreactive ve forms fc reactive of arginase in Neurospora crossa. J. Bact. 169, 5510-5517. Brown G. W. Jr and Cohen P. P. (1960) Comparative biochemistry of urea synthesis. is. 3--Activities of urea cycle enzymes in various higher vertebrate and lower vertebrates. Biochem. J. 75, 82-91. Chapula W., Clark J., Opliger ;r P. and Lavker R. (1970) Ammonia metabolism in rumen umen bacteria and mucosa from sheep fed soy proteinn and urea. J. Nutr. 100, 161-169. Chen P. C. and Broome J. D.• (1980) Mouse macrophage arginase. Pro& Soc. exp. Biol. ol. Med. 163, 354-359. Church D. (1971) Digestive Ph Physiology and Nutrition of Ruminants, Vol. 1, pp. 199-223. -223. O.S.U. Book Stores, Corvallis, OR. Cooper W. K. (1962) The infiue ifluence of term and ration composition on blood and rumen mucosal glutamate synthesis in sheep. M.S. Thesis, West Morgantown, VA.
Cornelius C. E. ~1 enzymology. In Clinical nimals (Edited by Kaneko Biochemistry t 5-236. Academic Press, J. I.), 3rd New York. Currie G. A. (l d macrophages kill tumor cells by relq se. Nature, Lond. 273, 758-759. Dahlig E., Porch Vlochnacka I. (1975) Purification and sort ' arginase from human lung• Acta Biochem. -85. De Ruitter H. a~ (1982) Activity of enzymes te cotyledons of developing of arginine me ~lant. Physiol., Bethesda 70, and germinatil 313-315. Emmanuel B. cycle enzymes in tissues (liv liver, rumen e rt, kidney, lung and spleen) of sheep (Ovi T. Biochem. Physiol• 65B, 693-697• Emm~ Emmanuel B. an • (1978) Studies on enzymes of nitrogen m nitrogen end products in developing ch 9mesticus) embryo• Comp. de~ Bio Biochem. Phys ~.89. Greenberg D. M es of urea cycle. In Enzymes Greet (Edited by Bo~ y H. and Myrback K.), 2nd (Ec edn, tdemic Press, New York• edr Vol. 4, Pt Hosh Hoshina S., Saur ~l orimato K. (1966) Ammonia metabolisn J. Dairy Sci. 49, 1523-1528. nia Hous H. L. (1 House tabolism. In Physiology of Ins,ects (Editec n M.), Vol. 2, pp. 769-813. Ac~ Academic Pre, Ide Y. (1969) Enzymatic activities in ruminal rt mucosa--II. Pre Presence and activities of L-glutamat utamate dehydrogenase in goats• Jap. J. Vet. Sci. 31, 99-103. Kona Konarsh L., Tomaszewski L., Colombo olombo J. P. and Terheggen H. G. (1985) Human salivary anc and arginase and its def deficiency in argininemia. J. clin. Chem. Chef, clin. Biochem. 23, 337-342. Kure] Kurelec B., Haarmeyer J. and Hill H. (1968) Uber funktiol tionen de Arginase, Urease und ad Ornth Ornthintranscarbamylase der der Pansenwand. 1. Mitteilung: Arg ianse- and Ureasefun funktion. Zbl. Vet. Med. 15, 460-46 460-469. Lowr O. H•, Rosebrough N. J., Farr A. L. and Randall Lowry A A.J. (1951) Protein measurement with wil the Folin phenol reagent• J. biol. Chem. 193, 265-275 265-275. Mora J., Martucelli J., Ortiz-Pineda J. and al Sobern G. (1965) The regulation of urea biosynthesis en thesis enzymes in vertebrate. Biochem. J. 96, 28-35. Nesheim M. C. and Garlich J. D. (It (1963) Studies on ornithine synthesis in relation to benzoic benzo acid excretion in domestic fowl. J. Nutr. 79, 311-317. Pace N. C., Buono A. and Simson H. S. (1980) Steady-state kinetic studies of arginase with an ir~tproved direct spectrophotometric assay. Analyt. Biochem. Bioche 109, 261-265. Porta R., Eposito C., Martin A. and Piera G. D. (1976) Arginase from human full-term placer flacenta. Biochem. J. 159, 579-583. Reyero C. and Doner F. (1975) Purification Puril of arginase from human leukemic lymphocytes and granulocytes: study of their physiochemical and kinetic kin properties. Eur. J. Biochem. 56, 137-147• Ruegg U. T. and Russell A. S. (1980) A rapid and sensitive assay for arginase. Analyt. Biochem. 102, 206-212. Snelman A., Aperia A. and Broberger O. ( M. (1979) Studies of renal urea cycle enzymes• II. Human Hu renal arginase activity and location of the adaptiv tptive changes of renal arginase in the protein-deprived rat. Scand. J. clin. lab. Invest. 39, 337-342• Spector E. B., Rice S. C. H., Moedj~ono S., Bernard B. properties of and Cederbum S. D. (1982) Biochemical Bioche arginase in human adult and fetal tis tissues. Biomed. J. 28, 165-175. ad Cederbum S. D. (1983) ginase in tissues of normal
Arginase distribution ients. Pediat. Res. 17, lltiple molecular forms iochem. Biophys. 260, mparison of biochemin streptozocin-induccd Biochem. Biophys. 274, L) Tissue and subcellurginine metabolism in • Res. Commun. 101,
Vanella N., lair Disilresto I. q content in diff~ development.. Van Elsen A. F. in human dir Commun. 62, Whanger P. D. a ties of ruminal Sci. 30, 944-9 Zamecka E. an arginaseinhut 258-266.
~isada A., Durso P. and ge activity and polyamine f rat brain during postnatal Vaples 34, 452-458. (1975) Arginas¢ isoenzymes ts. Biochem. biophys. Res. C. (1970) Enzymatic activist epithelial tissues. J. Dairy Z. (1988) Five forms of ,chem. Med. Metab. Biol. 39,
~TRIBUTION IN TISSUE', ANIMALS V[AHMOUD AMINLARI*
and
)MESTIC
TALEB VASI
ry, School of Veterinary Medicine, Shiraz
iraz, 71365 Iran
Received 6 March 1992; accepted 8 April 1 ofal A~tract--1. A new colorimetric method was usedI for determination determi domestic animals. 2. In all species studied liver was the richest source of arginase. 3. Significant differences were observed in the specific activity activi of argin~ 4. In all species, besides liver, kidney and brain also con contained signi 5. In the dog, in addition to the three organs mentioned aabove, lung, showed some arginase activity. in 6. In sheep and cattle significant arginase activity was observed c observed between epithelial and muscular layers of different parts 1z of dige 7. These results are discussed in terms of the possible role rol of argina
INTRODUCTION
rginase (L-arginine amidinohydrolase, EC 3.5.3.1) is terminal enzyme of the urea cycle which catalyzes thee hydrolysis of L-arginine into urea and L-ornithine (Greenber ;reenberg, 1960). An extraordinarily extensive literature ure is available on arginase and many aspects of this is enzyme from different sources have been studied, Besides :sides its involvement in ammonia detoxification whir hich occurs through the urea cycle, the role of ar ginase in other processes such as cell toxicity due to decreasin :creasing arginine in macrophage culture (Currie, 1971 ~78), inhibitory effects on lymphoma cells in vitro (Reyero and Doner, 1975; RLuggs u and Russell, 1980), and its elevated level in growmg wing tissues and tumors (Taylor and Stewart, 1981, and references therein) has been reported. F u r t h e rrmore, m o r e arginase plays a key role in the biosynthesis; of polyamines in brain ~es (Vanella et al., 1979). and other extrahepatic tissues This enzyme is widely distributed xibuted in nature, from bacteria to man (Greenberg,g, 1960; Cornelius, 1980; De Ruitter and Kollofel, 1982; 82; Borkovich, 1987, and references therein). Atthou gh the distribution and properties of arginase from many tissues of human (Dahlig et al., 1975; Reyeero and Doner, 1975; ~75; Borcic and Straus, Van Elsen and Leroy, 1975; 1976; Porta et al., 1976; Snelman et al., 1979; ~80; Spector et al., 1982, Baranczyk-huzma et al., 1980; 1983; Konarsh et al., 1985; Zamecka cka and ~amecka and Porembska, 1988) and laboratory animals mls (Chen and Broome, 1980; Brusdeilins et al., 1985 )85; Spolaris and Bond, 1988, 1989) have been extensiv, ensively studied, little is ~tribution of this enzyme known of the pattern of distributio~ in the tissues of domestic animals. stigation was to examine The purpose of this investi gtribution of arginase in and compare the tissue distributio~ Jlts of these studies will domestic animals. The results help to assess the role of ar ginase in different tissues [ n ee
*To whom correspondence should be a~
ent tissues of some n different species. arginase. nd skeletal muscle '
o differences were all species studied. tissues of animals.
of animals. ani We , shown that the activity of rhodanese rh rnoaanesc (~.t~ 2.5.1.1) ana md beta-mercaptopyru~t C vate sulfurtransferase (EC 2.8.1.2!), two enzymes invob involved in cyanide detoxification, is higher in rumen epith( fithelium than in liver (Aminla Aminlari et al., 1989; Amin Aminlari and Gilanpour, 1991). In the l present study we tl tried to find out whether suc such a pattern of distril distribution exists for arginase. This " paper also repor 3orts on the application of a new t method for argin~ inase which has been developed )ed il in this laboratory. This simple colorimetric method is based on the deten determination of the remaining arginine, al after its hydr( hydrolysis, by reaction with p-nitrophenyl p-nitt glyoxal (PNPG) (Aminlari, 1992). This method met allows rapid determination of arginase in a hlarge number of samples. METHODS MATERIALS AND METH
All chemicals were of analytical grade and were supplied by commercial sources, p-Nitrophenyl gl glyoxal (PNPG) was synthesized as described previously (Aminlari, 1992). Organs of sheep (Ovis aries), cattle (Bos 0 taurus), camel (Camelus dromedaries), were obtained from slaughtered animals. Post-mortem horse (Equus caballus ), donkey familiaris) samples were (Equus asinus), and dog (Canis familk obtained from Veterinary School hospifital. These samples were prepared by autopsy of animals, 0-2 0-~ hr after death. All samples, kept on ice, were transferred within w 45 min to the laboratory; tissues were separated, stripped stril from fat and extraneous materials, washed a few time,, with physiological few times saline and then blotted. Tissue extracts extracts were prepared by freezing 1 g of the sample in liquid nitrogen, nitre homogenizing pending the homogenate with a hand-homogenizer, and suspendi in 4 ml of 0.025 M sodium phosphate buffer, I pH 7.2. The min at 4000 g in an suspensions were centrifuged for 15 m ge. The supernatants MSE high-speed refrigerated centrifuge were used as the source of enzyme. The muscular and epithelial layers of different partsLsof stomach ston and small and large intestine were separated and treated tr, as described _1_ . . . . A __: . . . . . . . . . . . . . . . . .I L . . .i. t by the PNPG method lari, 1992) with some
MAHMOUDAM1NLARI and TALEB VASE(
at pH 9.5 was added e was kept at 37°C for ieved by adding 0.2 ml ntaining 0.1 M sodium m ascorbate, pH9.0. volume of the reaction t distilled water and the gainst a blank containg a Unicam Model SP tandard curve a molar cm was obtained,
Determination o f arginase activity To a 0.2 ml solution of 20 mM arginine at pH 9.5 was :lded 20 pl 10% MnCI 2 and the mixture was warmed in a ater bath at 37°C for 5 min. Incubation was started by :lding 10-/d tissue extract and holding the mixture at 37°C )r 20 min. These solutions were treated for color developtent as described above. The concentration of the remainLg arginine was determined using the molar extinction )efficient (see above). For each sample, a control solution as routinely included which contained all solutions except tat no arginine was present. This control was included in rder to eliminate any interference caused by the reaction of NPG with constituents in the serum (such as arginine sidues of proteins). The absorbance due to this control was tbtracted from the absorbance of respective samples before dculating the pmol arginine remaining. One unit of •ginase activity was defined as #mol arginine hydrolyses ~r min at 37°C and pH9.5 and was calculated from following equation: Unit activity =/~mol arginine/min = (AA4s0/2.6 x 103) x 104 × 1/T. InL the above equation AA4s0 is the difference of the ab)rbance at 480 nm of a solution of arginine in the absence sorbance an(ld presence of arginase, 2.6 x 103 is the arginine-equivalent molar olar extinction coefficient, 104 is the factor for converting
mol/1 arginine to and T is the inc
in a 10 ml reaction mixture, usually 20 min).
C. Protein deten Protein was d 0953).
the method o f Lowry et al.
,TS A r g i n i n e rea pyrophosphate p H 9.0 a n d 37
4PG in 0.1 mol/l s o d i u m s o d i u m a s c o r b a t e , at ce a c o l o r e d c o m p o u n d which absorbe at 480 n m with a m o l a r eextin x t i n c t i o n coef x 103 M / c m . T h e maxim m absorbal t i n e d after 3 0 m i n . T h e m uu m rreact eaction descr was used to d e t e r m i n e m a s e activil argin aracts. A f t e r a d d i t i o n o f a ssan ample conta e to a p r e - m i x e d arginine solut solution and i r 20 min, the r e m a i n i n g arginine was react with P N P G . T h e diffel difference in tl: ,~ o f a n arginine s o l u t i o n in the absen( ~ence o f arginase was ccorre o r r e l a t e d to t] ' arginase in the sample. T h e specific ginase in different tissues Th d o m e s t i c an vn in Tables 1 a n d 2. In o f de species ;r was f o u n d to be the all the tl fiche: source richest In the dog, the levels o f argin mase in kidney, brain, spleen, hhee a r t a t r i u m , h e a r t ventr ventricle, a n d skeletal muscle are silgnificantly higher t h a n in o t h e r tissues, except the liver ( P < 0.01) (Tab'~ 1). In all species studied,, kkid1 (Table i d n e y a n d b r a i n are two o( r g a n s w h i c h c o n t a i n significm aificant activity o f this enzyi~me as c o m p a r e d to o t h e r o r g aans r listed. In sheep a n d cattle, a significant level o f arginase is also 9resent in the r u m e n (Table 2). N o significant differprese ence was o b s e r v e d in the arginase c o n t e n t b e t w e e n the m rr u s c u l a r a n d epithelial layers rers o f different p a r t s o f
Table 1.. Mean (_+SD) of the specific activity of arginase in the crude extracts from different tissues of dog, horse and donkey "I~^L.I^
1
Tissues
ltm~--
/
t
or'~\
_$'.l.
. . . . .
:~-
Dog N=5
~--,1..1
....
e-.
Units/mg protein Horse N=2
Donkey N=4 0.37 (0.04)*t 0.06 (0.010):~ 0.02 (0.008) 0.02 (0.007) O.02 (O.O02) 0.06 (0.020):~ O.O2 (O.OIO) O.O2 (O.OO9)
Liver 0.41 (0.07)*'[" 0.49 (0.04)*t Kidney 0.08 (0.01):~ 0.06 (0.002):~ Heart ventricle 0.08 (0.040)J; 0.03 (0.003) Lung 0.05 (0.020)~ 0.03 (0.003) Spleen 0.06 (0.010);~ 0.01 (0.002) Brain 0.06 (0.020):~ 0.06 (0.010)~ Heart atrium 0.09 (0.040)* 0.03 (0.006) Skeletal muscle 0.08 (0.030)~: 0.04 (0.007) Cardiac region of stomach 0.02 (0.004) Pyloric region of stomach 0.02 (0.010) Glandular part of stomach 0.02 (0.008) 0.01 (0.004) Preventricular part of stomach -0.01 (0.006) 0.02 (0.010) Duodenum 0.02 (0.010) 0.01 (0.001) 0.02 (0.015) 0.01 (0.006) 0.02 (0.006) 0.01 (0.007) Ileum 0.01 (0.007) 0.01 (0.003) 0.01 (0.004) Jejunum Colon 0.02 (0.010) 0.01 (0.002) 0.02 (0.010) Cecum 0.02 (0.005) 0.01 (0.008) 0.02 (0.014) Rectum 0.02 (0,010) ND ND N = Number mber of animals studied. ND = Not determined. *Value of this tissue is significantly different from all other tissues (P < 0.01). tThis value alue in the horse is significantly different from that of donkey (P < 0.01). :[:In all species, val but not signifi dpt (P < 0.01). -
-
-
-
-
Jes)
Arginase distribution + SD) of the specificactivityof arginasc in th different tissues of sheep, cattle and camel Units/mg protein Cattle Sheep
Omasum Reticulum Rumen Abomasal fundus Abomasal pylore Duodenum lleum Jejunum Colon Rectum
N=5
N=5
0.42 (0.03)*?
0.4o (0.o4)*?
0.08 (0.006)~:
0.09 (0,009):~
0.05 (0.010) 0.02 (o.001) 0.02 (0.001)
0.03 (0.009) 0.o4 (0.008) 0.03 (0.0O7)
0.08 (0.02):~ 0.04 (0.009) 0.02 (0.006) O.O1 (0.006)
0.08 0.03 0.02 0.02
0.03 (0.010)
0.o4 (0.009)
0.05 (O.OlO)?:l: 0.02 (0.006)
0.06 (0.008)Ui 0.03 (0.008)
0.03 (0.004)
0.02 (0.006)
0.02 0.03 0.01 0.01 0.01
0.01 0.02 0.01 0.01 0.01
(0.008) (0.010) (0.003) (0.005) (0.006)
(0.010):~ (0.004) (0.008) (0.008)
(0.009) (0.005) (0,006) (0.007) (0.006)
N = Number of animals studied. *Value of this tissue is significantly different from all ," other tisst ?These values in sheep and cattle are significantlydiff different from th :[:In all species, values of these tissues are significantly significanl different t not significantly different from values beating the th( same letter
igestive systems of all animals studied (data not town). No intra- or inter-species differences were Observe(1 bserved in arginase activity of any other tissues of animals listed in Tables 1 and 2.
DISCUSSION
In this paper, we describe the application of a new me tethod for the determination of arginase in different tissues ssues of domestic animals. This method is simpler and ad more rapid than other available methods for argmase, and uses fewer chemicals. I this method the s. In micals. sample containing arginase is added to a premixed incubation mixture and color or formation is achieved by the addition of a single reagent, i.e. PNPG. This compound performs two fun( notions. It reacts with the remaining arginine in the reaction mixture and, in effect, stops the arginase activit ctivity. Furthermore, the reaction product of arginine Le with PNPG is colored and can be used to calculate late arginase activity as described in the Methods section. These features greatly simplify the method ~d for determination of arginase and allow the screenin ning of a large number of samples in a short period of tim, time. The PNPG method has enough sensitivity to be.~used to compare tissues with little arginase activity. The data presented in thi:s communication indicate that liver is the richest source',e of o f arginase. In liver, the level of arginase is at least one order of magnitude greater than most other tissu( rues. The role of arginase in the metabolic life of cells ~ells has generally been considered in terms of its function unction in the urea cycle, In mammals, the liver is the ot organ in which a full urea cycle is functional (Greenber erg, 1960). However, the metabolic function of arginase m extrahepatic tissues use in is still unclear. The presence ce of arginase in tissues athwav~ fnr di~na~ino af of organisms using other pathways nitrogen has been explained as a deletion or repression of other enz,~
from
,? )~ ) ) ) )~ ) ) ) )t ) ) ) ) ) ) ) 0.01). s, but
O.Ol).
cycle (Brown I IJIVV'Vll O a. l t t . l k . . U t l ~ t l , I y960; l/V, •Vl Mora et al., 1965). Our, data show that besides liver, some s( other tissues of domestic animals show significant activity of argin mase. In all animals, kidney and brain also show considerable arginase activity. The presence of argin mase in extrahepatic tissues mitght indicate that these tissues use arginase for purposes puq other than urea synthesis. It has been reported )orted that for chickens (Emmanuel and Gilanpour, 1978), certain insects tHou House, 1965) and cells in tissue culture cult~ (Nesheim and Garli Garlich, 1963), arginine is an essenti essential nutrient. Dietary arginine serves as the sole source sol of ornithine which, in turn, can be metabolized to either proline or glutamate. Arginase can therefor~ herefore function physiologically in the metabolism of arlginine to proline and/or glutamate. Ornithine is also alst important as a precursor for polyamine biosynthesi ,nthesis which is necessary for cell division and differenti~ fferentiation. Such functions might be significant in the brain bra (Vanella et aL, 1979). A high arginase level in br brains of domestic animals might be due to its role in this regard. Our data show the presence of ar!ginase in different parts of the digestive system of sheep, ~, cattle and levels No significant camels, albeit at very low levels. difference is observed in the level of arginase between the epithelial and the muscular latyers of different parts of the digestive system of r~ ruminants. As we have reported earlier, rumen epithelium shows considerable activity of cyanide-i :anide-metabolizing enzymes, rhodanese and beta-mercapt tptopyruvate sulfurtransferase (Aminlari et al., 1989, Aminlari and Gilanpour, 1991). Although the rm rumen of sheep and cattle contains some arginase (Table (Tab 2), it probably does not play a significant role in aammonia detoxification as compared with liver, In h ruminants, the degradation of nitrogenous comp, ?pounds by rumen microorganisms leads to the produc )roduction of high levels c~f ~ r n m c m i ~ in t h p rnm@n [tr~hnro (Church, 1971). Highly amonia assimilation are n the rumen to prevent
MAHMOUD AMINLARI and TALEBVASEG
o u r results a n d the gators (Emmanuel, anger and Church, ruminants, arginase are n o t the main dfication in the rum o n i a in the r u m e n dehydrogenase (EC 1969), glutamine pula et al., 1970; saminases ( C h a p u l a anger a n d Church, )70), m i g h t be involved in this mechanism. As liver the richest source o f arginase, it would be expected lat the availability o f a simple m e t h o d for assaying lis enzyme, such as the one described in this paper, :nders arginase a suitable candidate for liver ruction tests in domestic animals. This idea is u n d e r arrent investigation in this laboratory• cknowledgements--This research was financially sup~rted by Grant No. 68-VE-523-281 from the Research ouncil of Shiraz University. Part of the data presented in ds paper was taken from theses by P. Najmodini, • Khalesi and M. Abdolhouseinzadeh.
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