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Comparison of influence of 2-oxoglutarate and glutamate on arginase activities in liver and kidney-cortex of rabbit, Oryctolagus cuniculus
ISSN 0305-0491/96/$15.00 PI1 S0305-0491(96)00154-X
Comp. Biochem. Physiol. Vol. l15B, No. 3, pp. 393-398, 1996 Copyright © 1996 Elsevier Science Inc.
ELSEVIER
Comparison of Influence of 2-Oxoglutarate and Glutamate on Arginase Activities in Liver and Kidney-Cortex of Rabbit, Oryctolagus cuniculus Jolanta Pietkiewicz and Jadwiga Bryla INSTITUTE OF BIOCHEMISTRY, UNIVERSITY OF WARSAW, AL. ZWIRKI I WIGURY 93, 02"089 WARSAW, POLAND
ABSTRACT. The effect of branched-chain amino acids, 2-oxoglutarate, glutamate and lysine on arginase activity was studied in liver and kidney-cortex of rabbit. In contrast to liver, where arginase was localized in both cytosol and mitochondria, the enzyme activity in rabbit kidney-cortex was exclusively exhibited in the mitochondrial fraction and was not activated by Mn 2+ ions. The branched-chain amino acids (leucine, isoleucine and valine) significantly inhibited urea production in both kidney-cortex and liver Initochondria and in the liver cytosolic fraction, whereas lysine was less effective. In contrary, the urea production in intact and permeabilized kidney-cortex mitochondria was decreased by about 30-50% by glutamate and 2-oxoglutarate added at 2 mM concentrations, whereas arginase activities in both cytosolic and mitochondrial fractions of rabbit liver were not affected by these compounds in the presence and absence of Mn 2. ions. 2-Oxoglutarate appeared to be a poor noncompetitive inhibitor of kidney-cortex arginase (K, = 6.2 _+ 0.9 raM), whereas glutamate was the competitive one (K, = 1.5 _+ 0.1 mM). When methionine sulfoximine, an inhibitor of glutamine synthetase, was added to renal tubule suspension to accumulate glutamate inside mitochondria, the urea production was lower than that on the inhibition of glutamate generation by aminooxyacetate, an inhibitor of aminotransferases, despite an increase of ornithine content in the particulate fraction, indicating that kidney-cortex arginase might be affected by glutamate under physiological conditions. Copyright © 1996 Elsevier Science Inc. come mOCHEM PHYSIOL115B;3:393-398, 1996. KEY WORDS. Arginase, branched-chain amino acids, glutamate, kidney-cortex, liver, lysine, 2-oxoglutarate, rabbit INTRODUCTION Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is the terminal enzyme of the urea cycle catalysing the hydrolysis of L-arginine into urea and t-ornithine. Mammalian arginase is mostly found in liver (16), where all enzymes of the urea cycle are abundant, and is used as a mechanism for elimination of ammonium ion in ureotelic animals. However, in other tissues, including the kidney, the main function of arginase is the production of t-ornithine, a precursor of glutamate and proline (12,18) as well as polyamines such as putrescine, spermidine and spermine (37). Like rat liver arginase (8,11 ), the kidney enzyme has been found in both cytosolic and mitochondrial fractions of rat (28,31,34). The main form of enzyme in liver is the cytosolic arginase AI (11,16), whereas in the kidney the mitochondrial isozyme All occurs (5,36). Although the latter isoform exhibits some similar kinetic and physicochemical properties, it can Address reprint requests to: J. Bryla, Institute of Biochemistry, Al. Zwirki i
Wigury 93, 02-089 Warsaw, Poland. Tel. and Fax (4822} 23-20-46; e-mail [email protected]. Abbreviati,ns---AOA, aminooxyacetate; MSO, methionine sulfoximine. Received 19 December 1995; accepted 22 May 1996.
be distinguished from AI immunochemically, electrophoretically and biochemically (5,35,36). A number of amino acids have been found to inhibit the liver enzyme. The most powerful are ornithine and lysine (2,30), which were identified as competitive inhibitors. Although certain branched-chain amino acids such as leucine, isoleucine and valine have been reported as arginase inhibitors in liver (3), human heart (1) and kidney (5), the type of inhibitions varies from species to species. In the present investigation, we show that in contrast to arginase of cytosolic and mitochondrial liver fractions, the activity of kidney enzyme is inhibited by 2-oxoglutarate and glutamate, whereas lysine, leucine, isoleucine and valine decrease enzyme activities in both tissues studied.
MATERIALS A N D METHODS
Preparation and Incubation of Kidney Cortex Tubules Renal cortex tubules from rabbits (male, Termond strain, 2 - 3 kg body weight) were isolated as described previously (40). The dry weight of tubules was determined according to Krebs et al. (20). Isolated kidney tubules (about 10 mg dry wt) were incubated at 37°C in 2 inl of Krebs-Ringer bicarbonate buffer in 25-ml plastic Erlenmeyer flasks sealed
394
with rubber stoppers under an atmosphere of 95% 02 + 5% CO2. Arginine was added at 2 mM concentration. For the measurement of urea production, the reaction was stopped after 60 min of incubation by the addition of 35% perchloric acid (0.1 vol of suspension). Content of metabolites in particulate fraction of renal tubules was estimated according to Zuurendonk and Tager (41) with minor modifications (4).
J. Pietkiewicz and J. Bryta
drial suspension through the silicone oil layer (19). After supernatant removal, tubes were washed three times with distilled water and the bottom perchloric acid layers were collected, neutralized and used to assay 2-oxoglutarate and glutamate, where indicated.
Assays of Metabolites Mitochondrial Preparations and Incubations Mitochondria were prepared from kidney-cortex and liver of Termond strain male rabbits (about 2-3 kg in weight) by a minor modification of the method described previously (33). The isolation medium contained 225 mM mannitol, 75 mM sucrose, 5 mM MOPS, 0.1 mM EDTA and 0.1% bovine serum albumin at pH 7.2. The final wash and suspension of mitochondria were made with 0.3 M mannitol. Treatment of isolated mitochondria with toluene to increase membrane permeability was done according to Matlib et al. (26). To remove enzymes of cytosolic fraction, liver mitochondria were washed with 150 mM KCI as described (8). Mitochondria (about 2-3 mg/ml) were incubated at 30°C in the reaction mixture containing 50 mM Tris-HCl (pH 7.6), 150 mM mannitol, 10 mM potassium phosphate buffer (pH 7.6), 5 mM arginine and 0.2 mM aminooxyacetate (AOA) to inhibit ornithine aminotransferase. The reactions were started by the addition of mitochondrial suspension to the reaction medium. When the effect of Mn 2+ ions on arginase activity was studied, mitochondria were preincubated for 5 min with 5 mM MnC12. For measurement of the total content of metabolites in the reaction mixture, 1-ml samples were withdrawn from the reaction mixture at 10-min intervals up to 30 rain and the protein was precipitated with 1/10 volume of 35% (v/v) perchloric acid.
Cytosol Preparation and Incubation Both liver and kidney-cortex were placed in 5-fold volumes of 0.3 M mannitol and homogenized in Potter-Elvehjem homogenizer. Homogenate was centrifuged at 18,000 g for 10 min. The supematant (5-15 mg of protein/ml) was used for arginase activity determination. The reaction was started by the addition of cytosol (0.1-0.3 mg/ml) to the reaction mixture containing 50 mM Tris-HCl (pH 7.6), 150 mM mannitol and 5 mM arginine. Samples were removed from the reaction medium at 2-min periods up to 6 rain of incubation and deproteinized with 0.1 volume of 35% perchloric acid.
Determination of Intramitochondrial 2.0xoglutarate and Glutamate Levels Accumulation of 2-oxoglutarate and glutamate in mitochondria was determined after centrifugation of mitochon-
Urea was measured either colorimetrically in the presence of 1-phenyl-l,2-propanedion-2-oxime (9) or by enzymatic determination of ammonia after the treatment of samples with urease (10). 2-Oxoglutarate was measured fluorimetrically with the use of glutamate dehydrogenase (39), whereas amino acids were determined by HPLC after derivatization of samples with 4-dimethylaminoazobenzene-4'sulfonyl chloride (7). Mitochondrial and cytosolic protein was determined by the biuret method (23) with bovine serum albumin as standard.
Expression of Results Data shown are means + SEM for three to four separate experiments. K~ values were calculated as described previously (14). The statistical significance of differences was estimated by Student's t-test.
RESULTS A N D DISCUSSION Effect of Branched.Chain Amino Acids, Lysine, 2.0xoglutarate and Glutamate on Arginase Activities in Mitochondr/a/and Cytosolic Fractions of Liver and Kidney.Cortex Because arginases are known to be distributed between the mitochondrial and cytosolic fractions of mammalian liver cells (11,16), the enzyme activity was determined in both cytosol and mitochondria washed with 150 mM KCI to remove the cytosolic enzyme attached to the outer mitochondrial membrane (8). Moreover, the arginase activity was measured in permeabilized mitochondria to abolish barrier for the transport of substrates and effectors of the enzyme. The rate of urea production was linear in time at least up to 30 min of incubation under all conditions studied. In agreement with a previous report (38), the activity of arginase in the rabbit liver cytosol exceeded markedly that determined in mitochondrial fractions isolated from either liver or kidney-cortex (Table 1), whereas arginase activity in the kidney-cortex cytosolic fraction was hardly detectable (data not shown), confirming observations reported for extrahepatic arginases (13,18,34). Moreover, in contrast to previous reports (6,21,22,30) but in agreement with Kaysen and Strecker (18), the activity of arginase extracted from rabbit kidney-cortex was not increased by incubation with Mn 2+. The activity of liver arginase is known to be inhibited by various amino acids such as branched-chain amino acids (5,27), ornithine (29,35), lysine (27,29,35) and proline
Arginases in Liver and Kidney of Rabbit
395
TABLE 1. Influence of 2.oxoglutarate and several amino acids on liver and kidney-cortex arginases Arginase activity ( n m o l / m i n per mg protein) Liver cytosol Additions
None 2-Oxoglutarate L-Glutamate L-Leucine L-Isoleucine L-Valine L-Lysine
- M n z+ 139.0 139.0 128.2 54.6 37.3 37.3 104.5
-+ 8.9 _+ 8.9 -+ 7.2 --+ 0.3* --+0.7¢ --+0.7¢ --+ 8.3***
+ M n 2+ 392.5 397.0 388.7 141.8 71.3 92.2 330.6
Liver mitochondria
-+ 10.0 + 7.4 -+ 2.7 + 7.9¢ --+ 11.7"* + 5.6*** + 6.0**
14.2 14.5 14.0 8.3 4.3 6.3 11.4
Renal mitochondria
-+ 1.1 -+ 0.9 -+ 0.6 +- 0.2t --+0.1¢ --+ 0.2* --+ 1.0t
2.5 1.9 1.5 1.7 1.1 1.3 1.9
+ 0.1 -+ OAt -+ 0.15 + 0.11" + 0.1" --+ 0.1" --+ 0.1"
Cytosol and permeabilized mitochondria were incubated in the reaction mixture described in Materials and Methods. The reactions were started with 5 mM arginine after a 5-rain preincubation of mitochondria with 2-oxoglutarate, amino acids and MnCI2 added at 2-, 2- and 5-raM concentrations, respectively, where indicated. Differences are significant for the inhibitory effects of compounds studied on arginase activity vs the corresponding controls at "~P < 0.05, $P < 0.02, *P < 0.01, **P < 0.005, ***P < 0.001, where indicated.
(5,18,29). Arginase extracted from the whole rat kidney tissue has also been observed to be sensitive to added amino acids, proline being a stronger inhibitor than branchedchain amino acids (5) and lysine being a stronger inhibitor than omithine (11,18). As shown in Table 1, branchedchain amino acids inhibited arginase activity by about 6 0 80% in both rabbit liver cytosol and mitochondria, whereas they decreased activity of renal enzyme in a smaller extent. L-Lysine was less effective than branched-chain amino acids. According to Spector et al. (35), K~ values for rat kidney arginase inhibition by lysine and ornithine were equal to 20 and 9 mM, respectively. L-Lysine was considered as a competitive inhibitor of rat liver arginase with Kt equal to 0.93 mM (30). However, in kidney-collecting ducts in vitro, the inhibition obtained with lysine seemed to result from competition between lysine and arginine for cell uptake via a c o m m o n membrane carrier, whereas the inhibition induced by branched-chain amino acids corresponded to an effect on arginase activity itself (24). The effect of isoleucine on liver and kidney-cortex arginase activities of the mitochondrial fractions of rabbit is presented in Fig. 1. This amino acid appeared to be a noncompetitive inhibitor of arginase in both liver and kidneycortex mitochondria with K~ equal to 0.9 + 0.1 and 1.2 -+ 0.1 mM, respectively, confirming observations for sheep liver and rat kidney (5,29). However, isoleucine has also been reported as a competitive inhibitor of purified arginase of bovine liver with K~ equal to 0.2 mM (27) and rat liver (5). Both 2-oxoglutarate and glutamate did not affect arginase activity in liver cytosol incubated without or with 5 mM Mn 2+ and in permeabilized liver mitochondria (cf. Table 1). In contrary, they diminished by about 3 0 - 4 0 % the rate of urea production in kidney-cortex toluene-treated mitochondria. However, there was no effect of malate, succinate, pyruvate, phosphoenolpyruvate, oxaloacetate, aspartate, alanine, proline and glutamine on arginase activity in permeabilized kidney-cortex mitochondria when added at as high as 5 mM concentrations (data not shown).
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FIG. 1. The effect of isoleucine on arginase activity in liver (A) and kidney-cortex (B) permeabilized mitochondria. Incubations were carried out in the presence of various arginine concentrations without ( 0 Q) and either with 2 mM ([] rT) or 5 mM ( b • ) isoleucine. Values shown are means -+ SEM for three separate experiments.
396
J. Pietkiewicz and J. Bryla
TABLE 2. Influence of intramitochondrially accumulated 2-oxoglutarate and glutamate on arginase activity in kidney-cortex mitochondria Arginase activity (nmol/min per Additions
None None 2-Oxoglutarate L-Glutamate
Rotenone
+ + +
2.Oxoglutarate (nmol/mg protein)
mg protein)
1.88 1.83 1.57 0.83 1.70 0.98
+ 0.09 + 0.08 -+ 0.15 * _+ 0.10"** + 0.26 -+ 0.23**
Glutamate (nmol/mg protein)
0.11 _+ 0.04
0.55 -+ 0.0t
0.11 0.25 0.13 0.12
1.05 + 0.01"** 2.45 -+ 0.60?
_+ 0.03 _+ 0.04* _+ 0.04 _+ 0.04
Arginine, 2-oxoglutarate, glutamine and rotenone were added at 5-raM, 0.5-mM, l-mM and 1-btM concentrations, respectively, where indicated. Intact mitochondria were incubated for 20 min with 0.2 mM AOA and centrifuged through the silicone oil. Urea production was measured in the supernatant, whereas 2-oxoglutarate and glutamate were determined in the acid extract of the mitochondrial pellet beneath the oil. Significant differences between values determined with either 2-oxoglutarate or L-glutaminevs corresponding controls are noted: tP < 0.05, *P < 0.02, **P < 0.01, ***P < 0.001.
To establish effects of intramitochondrially accumulated 2-oxoglutarate and glutamate on arginase activity, we separated intact kidney-cortex mitochondria from the reaction medium after the incubation in the absence and presence of rotenone (i.e., under conditions differentiating the rate of oxidation of metabolites studied). 2-Oxoglutarate was added at 0.5 mM concentrations to the reaction medium, whereas glutamate was generated from 1 mM glutamine via the reaction catalysed by glutaminase. As shown in Table 2, rotenone did not affect arginase activity in the absence of 2-oxoglutarate and glutamine in the reaction medium. Endogenous level of intramitochondrial 2-oxoglutarate was similar to that found in respiring mitochondria incubated with this compound, whereas it was 2-fold higher in rotenone-treated mitochondria, resulting in an inhibition of arginase activity by about 55%. Similarly, in the presence of glutamine, arginase activity was diminished by about 50% in rotenone-treated mitochondria, exhibiting 2-fold higher intramitochondrial glutamate level in comparison with that determined in respiring mitochondria. Because the intramitochondrial level of 2-oxoglutarate remained unchanged under both conditions studied, the inhibition of arginase activity in mitochondria incubated in the presence of glutamine and rotenone was probably due to the action of glutamate accumulated inside mitochondria. As shown in Figs. 1B and 2, K,,, value estimated for arginine in kidney-cortex toluene-treated mitochondria was higher than that reported for rat hepatic mitochondrial arginase (8) or rabbit liver cytosol enzyme (38) and was equal to 14.7 + 0.7 mM (as determined for eight separate experiments). It did not alter on the addition of 2-oxoglutarate to the incubation medium (14.2 -+ 0.9 mM, as determined for four separate experiments). In view of the kinetic analysis of data in the presence and absence of 2-oxoglutarate (Fig. 2A), it was possible to conclude that 2-oxoglutarate was a rather poor noncompetitive inhibitor of renal mitochondrial arginase with K~ value equal to 6.2 -+ 0.9 mM (as calculated for four separate experiments). Moreover, it was surprising that the negatively charged L-glutamate appeared
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FIG. 2. The effect of 2-oxoglutarate (A) and glutamate (B) on arginase activity in kidney-cortex permeabilized mitochondria. Incubations were carried out in the presence of various arginine concentrations without (O O) and either with 2 mM (In [3) or 5 mM (A i , ) 2-oxoglutarate and glutamate, respectively, as indicated. Values shown are means -- SEM for three separate experiments.
Arginases in Liver and Kidney of Rabbit
397
TABLE 3. Effect of AOA on the particulate levels of glutamate and ornithine and the urea production in isolated renal tubules incubated in the presence of arginine and MSO
AOA
Levels in the particulate fraction (nmol/mg dry weight) Glutamate Ornithine
+
7.7 + 0.3 0.8 _+- 0.1"
6.6 _+ 0.1 12.4 -+ 1.2"
Urea production (nmol/h per mg dry weight)
73.5 + 0.3 93.9 -+ 3.6*
Kidney-cortex tubules were incubated in the presence of 5 mM MSO either without or with 2 mM AOA. After 60 min of incubation, tubules were treated with digitonin and centrifuged as described in Materials and Methods. Significant differences between values measured with AOA against the corresponding ones determined without AOA are noted: *P < 0.01. to be a competitive inhibitor of renal mitochondrial arginase (Fig. 2B), with K, equal to 1.5 -+ 0.1 mM, particularly because the substrate t-arginine carries a positive charge at physiological pH.
Urea Production in Isolated Kidney-Cortex Tubules of Rabbit Under Conditions Differentiating Intracellular Glutamate Accumulation Because kidney cortex mitochondria exhibit omithine aminotransferase (15), omithine derived from arginine via the arginase reaction may react with 2-oxoglutarate, forming glutamate and glutamate-ysemialdehyde. Thus, glutamate may be considered as feedback inhibitor of rabbit kidneycortex arginase. To check whether the inhibitory effect of glutamate on kidney cortex arginase activity may be of physiological importance, we measured urea production in isolated rabbit renal tubules incubated with arginine and methionine sulfoximine (MSO), an inhibitor of glutamine synthetase (32), because M S O is known to elevate glutamate content in rabbit renal tubules (25). As shown in Table 3, glutamate was also accumulated in the particulate fraction isolated from renal tubules after digitonin treatment and centrifugation (41). With the addition of A O A , an inhibitor of aminotransferases (17), a marked decrease of glutamate generation via the reaction catalysed by ornithine aminotransferase resulted in a decline of glutamate level in the particulate fraction containing mitochondria accompanied by an enhancement of urea production for about 30%, despite 2-fold increase of content of ornithine, another inhibitor of arginase (35). In view of observations that levels of 2-oxoglutarate in the particulate fraction of rabbit renal tubules were low and varied between 0.05 and 0.15/amol/g dry weight under various experimental conditions (25) and glutamate concentration in the particulate fraction of rabbit renal tubules could reach 9 / l m o l / g tubule dry weight (25), it is likely that glutamate might inhibit the arginase activity in kidney-cortex tubules under physiological conditions. In summary, the data are consistent with the view that
despite sharing many similar physicochemical features, the hepatic and renal arginases are significantly different with respect to their regulatory properties. Both 2-oxoglutarate and glutamate appear to be inhibitors of rabbit renal arginase with no influence on the liver mitochondrial and cytosolic enzymes. We thank Mr. Tadeusz Lietz for performance of experiments with the use of rabbit renal tubules and Mr. Robert Jarzyna for assistance in the preparation of the manuscript. Technical assistance of Miss B. D~browska is acknowledged. This investigation was supported by the State Committee fi~r Scientific Research (Project 6 PO4A 024 08).
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Comp. Biochem. Physiol. Vol. l15B, No. 3, pp. 393-398, 1996 Copyright © 1996 Elsevier Science Inc.
ELSEVIER
Comparison of Influence of 2-Oxoglutarate and Glutamate on Arginase Activities in Liver and Kidney-Cortex of Rabbit, Oryctolagus cuniculus Jolanta Pietkiewicz and Jadwiga Bryla INSTITUTE OF BIOCHEMISTRY, UNIVERSITY OF WARSAW, AL. ZWIRKI I WIGURY 93, 02"089 WARSAW, POLAND
ABSTRACT. The effect of branched-chain amino acids, 2-oxoglutarate, glutamate and lysine on arginase activity was studied in liver and kidney-cortex of rabbit. In contrast to liver, where arginase was localized in both cytosol and mitochondria, the enzyme activity in rabbit kidney-cortex was exclusively exhibited in the mitochondrial fraction and was not activated by Mn 2+ ions. The branched-chain amino acids (leucine, isoleucine and valine) significantly inhibited urea production in both kidney-cortex and liver Initochondria and in the liver cytosolic fraction, whereas lysine was less effective. In contrary, the urea production in intact and permeabilized kidney-cortex mitochondria was decreased by about 30-50% by glutamate and 2-oxoglutarate added at 2 mM concentrations, whereas arginase activities in both cytosolic and mitochondrial fractions of rabbit liver were not affected by these compounds in the presence and absence of Mn 2. ions. 2-Oxoglutarate appeared to be a poor noncompetitive inhibitor of kidney-cortex arginase (K, = 6.2 _+ 0.9 raM), whereas glutamate was the competitive one (K, = 1.5 _+ 0.1 mM). When methionine sulfoximine, an inhibitor of glutamine synthetase, was added to renal tubule suspension to accumulate glutamate inside mitochondria, the urea production was lower than that on the inhibition of glutamate generation by aminooxyacetate, an inhibitor of aminotransferases, despite an increase of ornithine content in the particulate fraction, indicating that kidney-cortex arginase might be affected by glutamate under physiological conditions. Copyright © 1996 Elsevier Science Inc. come mOCHEM PHYSIOL115B;3:393-398, 1996. KEY WORDS. Arginase, branched-chain amino acids, glutamate, kidney-cortex, liver, lysine, 2-oxoglutarate, rabbit INTRODUCTION Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is the terminal enzyme of the urea cycle catalysing the hydrolysis of L-arginine into urea and t-ornithine. Mammalian arginase is mostly found in liver (16), where all enzymes of the urea cycle are abundant, and is used as a mechanism for elimination of ammonium ion in ureotelic animals. However, in other tissues, including the kidney, the main function of arginase is the production of t-ornithine, a precursor of glutamate and proline (12,18) as well as polyamines such as putrescine, spermidine and spermine (37). Like rat liver arginase (8,11 ), the kidney enzyme has been found in both cytosolic and mitochondrial fractions of rat (28,31,34). The main form of enzyme in liver is the cytosolic arginase AI (11,16), whereas in the kidney the mitochondrial isozyme All occurs (5,36). Although the latter isoform exhibits some similar kinetic and physicochemical properties, it can Address reprint requests to: J. Bryla, Institute of Biochemistry, Al. Zwirki i
Wigury 93, 02-089 Warsaw, Poland. Tel. and Fax (4822} 23-20-46; e-mail [email protected]. Abbreviati,ns---AOA, aminooxyacetate; MSO, methionine sulfoximine. Received 19 December 1995; accepted 22 May 1996.
be distinguished from AI immunochemically, electrophoretically and biochemically (5,35,36). A number of amino acids have been found to inhibit the liver enzyme. The most powerful are ornithine and lysine (2,30), which were identified as competitive inhibitors. Although certain branched-chain amino acids such as leucine, isoleucine and valine have been reported as arginase inhibitors in liver (3), human heart (1) and kidney (5), the type of inhibitions varies from species to species. In the present investigation, we show that in contrast to arginase of cytosolic and mitochondrial liver fractions, the activity of kidney enzyme is inhibited by 2-oxoglutarate and glutamate, whereas lysine, leucine, isoleucine and valine decrease enzyme activities in both tissues studied.
MATERIALS A N D METHODS
Preparation and Incubation of Kidney Cortex Tubules Renal cortex tubules from rabbits (male, Termond strain, 2 - 3 kg body weight) were isolated as described previously (40). The dry weight of tubules was determined according to Krebs et al. (20). Isolated kidney tubules (about 10 mg dry wt) were incubated at 37°C in 2 inl of Krebs-Ringer bicarbonate buffer in 25-ml plastic Erlenmeyer flasks sealed
394
with rubber stoppers under an atmosphere of 95% 02 + 5% CO2. Arginine was added at 2 mM concentration. For the measurement of urea production, the reaction was stopped after 60 min of incubation by the addition of 35% perchloric acid (0.1 vol of suspension). Content of metabolites in particulate fraction of renal tubules was estimated according to Zuurendonk and Tager (41) with minor modifications (4).
J. Pietkiewicz and J. Bryta
drial suspension through the silicone oil layer (19). After supernatant removal, tubes were washed three times with distilled water and the bottom perchloric acid layers were collected, neutralized and used to assay 2-oxoglutarate and glutamate, where indicated.
Assays of Metabolites Mitochondrial Preparations and Incubations Mitochondria were prepared from kidney-cortex and liver of Termond strain male rabbits (about 2-3 kg in weight) by a minor modification of the method described previously (33). The isolation medium contained 225 mM mannitol, 75 mM sucrose, 5 mM MOPS, 0.1 mM EDTA and 0.1% bovine serum albumin at pH 7.2. The final wash and suspension of mitochondria were made with 0.3 M mannitol. Treatment of isolated mitochondria with toluene to increase membrane permeability was done according to Matlib et al. (26). To remove enzymes of cytosolic fraction, liver mitochondria were washed with 150 mM KCI as described (8). Mitochondria (about 2-3 mg/ml) were incubated at 30°C in the reaction mixture containing 50 mM Tris-HCl (pH 7.6), 150 mM mannitol, 10 mM potassium phosphate buffer (pH 7.6), 5 mM arginine and 0.2 mM aminooxyacetate (AOA) to inhibit ornithine aminotransferase. The reactions were started by the addition of mitochondrial suspension to the reaction medium. When the effect of Mn 2+ ions on arginase activity was studied, mitochondria were preincubated for 5 min with 5 mM MnC12. For measurement of the total content of metabolites in the reaction mixture, 1-ml samples were withdrawn from the reaction mixture at 10-min intervals up to 30 rain and the protein was precipitated with 1/10 volume of 35% (v/v) perchloric acid.
Cytosol Preparation and Incubation Both liver and kidney-cortex were placed in 5-fold volumes of 0.3 M mannitol and homogenized in Potter-Elvehjem homogenizer. Homogenate was centrifuged at 18,000 g for 10 min. The supematant (5-15 mg of protein/ml) was used for arginase activity determination. The reaction was started by the addition of cytosol (0.1-0.3 mg/ml) to the reaction mixture containing 50 mM Tris-HCl (pH 7.6), 150 mM mannitol and 5 mM arginine. Samples were removed from the reaction medium at 2-min periods up to 6 rain of incubation and deproteinized with 0.1 volume of 35% perchloric acid.
Determination of Intramitochondrial 2.0xoglutarate and Glutamate Levels Accumulation of 2-oxoglutarate and glutamate in mitochondria was determined after centrifugation of mitochon-
Urea was measured either colorimetrically in the presence of 1-phenyl-l,2-propanedion-2-oxime (9) or by enzymatic determination of ammonia after the treatment of samples with urease (10). 2-Oxoglutarate was measured fluorimetrically with the use of glutamate dehydrogenase (39), whereas amino acids were determined by HPLC after derivatization of samples with 4-dimethylaminoazobenzene-4'sulfonyl chloride (7). Mitochondrial and cytosolic protein was determined by the biuret method (23) with bovine serum albumin as standard.
Expression of Results Data shown are means + SEM for three to four separate experiments. K~ values were calculated as described previously (14). The statistical significance of differences was estimated by Student's t-test.
RESULTS A N D DISCUSSION Effect of Branched.Chain Amino Acids, Lysine, 2.0xoglutarate and Glutamate on Arginase Activities in Mitochondr/a/and Cytosolic Fractions of Liver and Kidney.Cortex Because arginases are known to be distributed between the mitochondrial and cytosolic fractions of mammalian liver cells (11,16), the enzyme activity was determined in both cytosol and mitochondria washed with 150 mM KCI to remove the cytosolic enzyme attached to the outer mitochondrial membrane (8). Moreover, the arginase activity was measured in permeabilized mitochondria to abolish barrier for the transport of substrates and effectors of the enzyme. The rate of urea production was linear in time at least up to 30 min of incubation under all conditions studied. In agreement with a previous report (38), the activity of arginase in the rabbit liver cytosol exceeded markedly that determined in mitochondrial fractions isolated from either liver or kidney-cortex (Table 1), whereas arginase activity in the kidney-cortex cytosolic fraction was hardly detectable (data not shown), confirming observations reported for extrahepatic arginases (13,18,34). Moreover, in contrast to previous reports (6,21,22,30) but in agreement with Kaysen and Strecker (18), the activity of arginase extracted from rabbit kidney-cortex was not increased by incubation with Mn 2+. The activity of liver arginase is known to be inhibited by various amino acids such as branched-chain amino acids (5,27), ornithine (29,35), lysine (27,29,35) and proline
Arginases in Liver and Kidney of Rabbit
395
TABLE 1. Influence of 2.oxoglutarate and several amino acids on liver and kidney-cortex arginases Arginase activity ( n m o l / m i n per mg protein) Liver cytosol Additions
None 2-Oxoglutarate L-Glutamate L-Leucine L-Isoleucine L-Valine L-Lysine
- M n z+ 139.0 139.0 128.2 54.6 37.3 37.3 104.5
-+ 8.9 _+ 8.9 -+ 7.2 --+ 0.3* --+0.7¢ --+0.7¢ --+ 8.3***
+ M n 2+ 392.5 397.0 388.7 141.8 71.3 92.2 330.6
Liver mitochondria
-+ 10.0 + 7.4 -+ 2.7 + 7.9¢ --+ 11.7"* + 5.6*** + 6.0**
14.2 14.5 14.0 8.3 4.3 6.3 11.4
Renal mitochondria
-+ 1.1 -+ 0.9 -+ 0.6 +- 0.2t --+0.1¢ --+ 0.2* --+ 1.0t
2.5 1.9 1.5 1.7 1.1 1.3 1.9
+ 0.1 -+ OAt -+ 0.15 + 0.11" + 0.1" --+ 0.1" --+ 0.1"
Cytosol and permeabilized mitochondria were incubated in the reaction mixture described in Materials and Methods. The reactions were started with 5 mM arginine after a 5-rain preincubation of mitochondria with 2-oxoglutarate, amino acids and MnCI2 added at 2-, 2- and 5-raM concentrations, respectively, where indicated. Differences are significant for the inhibitory effects of compounds studied on arginase activity vs the corresponding controls at "~P < 0.05, $P < 0.02, *P < 0.01, **P < 0.005, ***P < 0.001, where indicated.
(5,18,29). Arginase extracted from the whole rat kidney tissue has also been observed to be sensitive to added amino acids, proline being a stronger inhibitor than branchedchain amino acids (5) and lysine being a stronger inhibitor than omithine (11,18). As shown in Table 1, branchedchain amino acids inhibited arginase activity by about 6 0 80% in both rabbit liver cytosol and mitochondria, whereas they decreased activity of renal enzyme in a smaller extent. L-Lysine was less effective than branched-chain amino acids. According to Spector et al. (35), K~ values for rat kidney arginase inhibition by lysine and ornithine were equal to 20 and 9 mM, respectively. L-Lysine was considered as a competitive inhibitor of rat liver arginase with Kt equal to 0.93 mM (30). However, in kidney-collecting ducts in vitro, the inhibition obtained with lysine seemed to result from competition between lysine and arginine for cell uptake via a c o m m o n membrane carrier, whereas the inhibition induced by branched-chain amino acids corresponded to an effect on arginase activity itself (24). The effect of isoleucine on liver and kidney-cortex arginase activities of the mitochondrial fractions of rabbit is presented in Fig. 1. This amino acid appeared to be a noncompetitive inhibitor of arginase in both liver and kidneycortex mitochondria with K~ equal to 0.9 + 0.1 and 1.2 -+ 0.1 mM, respectively, confirming observations for sheep liver and rat kidney (5,29). However, isoleucine has also been reported as a competitive inhibitor of purified arginase of bovine liver with K~ equal to 0.2 mM (27) and rat liver (5). Both 2-oxoglutarate and glutamate did not affect arginase activity in liver cytosol incubated without or with 5 mM Mn 2+ and in permeabilized liver mitochondria (cf. Table 1). In contrary, they diminished by about 3 0 - 4 0 % the rate of urea production in kidney-cortex toluene-treated mitochondria. However, there was no effect of malate, succinate, pyruvate, phosphoenolpyruvate, oxaloacetate, aspartate, alanine, proline and glutamine on arginase activity in permeabilized kidney-cortex mitochondria when added at as high as 5 mM concentrations (data not shown).
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FIG. 1. The effect of isoleucine on arginase activity in liver (A) and kidney-cortex (B) permeabilized mitochondria. Incubations were carried out in the presence of various arginine concentrations without ( 0 Q) and either with 2 mM ([] rT) or 5 mM ( b • ) isoleucine. Values shown are means -+ SEM for three separate experiments.
396
J. Pietkiewicz and J. Bryla
TABLE 2. Influence of intramitochondrially accumulated 2-oxoglutarate and glutamate on arginase activity in kidney-cortex mitochondria Arginase activity (nmol/min per Additions
None None 2-Oxoglutarate L-Glutamate
Rotenone
+ + +
2.Oxoglutarate (nmol/mg protein)
mg protein)
1.88 1.83 1.57 0.83 1.70 0.98
+ 0.09 + 0.08 -+ 0.15 * _+ 0.10"** + 0.26 -+ 0.23**
Glutamate (nmol/mg protein)
0.11 _+ 0.04
0.55 -+ 0.0t
0.11 0.25 0.13 0.12
1.05 + 0.01"** 2.45 -+ 0.60?
_+ 0.03 _+ 0.04* _+ 0.04 _+ 0.04
Arginine, 2-oxoglutarate, glutamine and rotenone were added at 5-raM, 0.5-mM, l-mM and 1-btM concentrations, respectively, where indicated. Intact mitochondria were incubated for 20 min with 0.2 mM AOA and centrifuged through the silicone oil. Urea production was measured in the supernatant, whereas 2-oxoglutarate and glutamate were determined in the acid extract of the mitochondrial pellet beneath the oil. Significant differences between values determined with either 2-oxoglutarate or L-glutaminevs corresponding controls are noted: tP < 0.05, *P < 0.02, **P < 0.01, ***P < 0.001.
To establish effects of intramitochondrially accumulated 2-oxoglutarate and glutamate on arginase activity, we separated intact kidney-cortex mitochondria from the reaction medium after the incubation in the absence and presence of rotenone (i.e., under conditions differentiating the rate of oxidation of metabolites studied). 2-Oxoglutarate was added at 0.5 mM concentrations to the reaction medium, whereas glutamate was generated from 1 mM glutamine via the reaction catalysed by glutaminase. As shown in Table 2, rotenone did not affect arginase activity in the absence of 2-oxoglutarate and glutamine in the reaction medium. Endogenous level of intramitochondrial 2-oxoglutarate was similar to that found in respiring mitochondria incubated with this compound, whereas it was 2-fold higher in rotenone-treated mitochondria, resulting in an inhibition of arginase activity by about 55%. Similarly, in the presence of glutamine, arginase activity was diminished by about 50% in rotenone-treated mitochondria, exhibiting 2-fold higher intramitochondrial glutamate level in comparison with that determined in respiring mitochondria. Because the intramitochondrial level of 2-oxoglutarate remained unchanged under both conditions studied, the inhibition of arginase activity in mitochondria incubated in the presence of glutamine and rotenone was probably due to the action of glutamate accumulated inside mitochondria. As shown in Figs. 1B and 2, K,,, value estimated for arginine in kidney-cortex toluene-treated mitochondria was higher than that reported for rat hepatic mitochondrial arginase (8) or rabbit liver cytosol enzyme (38) and was equal to 14.7 + 0.7 mM (as determined for eight separate experiments). It did not alter on the addition of 2-oxoglutarate to the incubation medium (14.2 -+ 0.9 mM, as determined for four separate experiments). In view of the kinetic analysis of data in the presence and absence of 2-oxoglutarate (Fig. 2A), it was possible to conclude that 2-oxoglutarate was a rather poor noncompetitive inhibitor of renal mitochondrial arginase with K~ value equal to 6.2 -+ 0.9 mM (as calculated for four separate experiments). Moreover, it was surprising that the negatively charged L-glutamate appeared
]4-
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FIG. 2. The effect of 2-oxoglutarate (A) and glutamate (B) on arginase activity in kidney-cortex permeabilized mitochondria. Incubations were carried out in the presence of various arginine concentrations without (O O) and either with 2 mM (In [3) or 5 mM (A i , ) 2-oxoglutarate and glutamate, respectively, as indicated. Values shown are means -- SEM for three separate experiments.
Arginases in Liver and Kidney of Rabbit
397
TABLE 3. Effect of AOA on the particulate levels of glutamate and ornithine and the urea production in isolated renal tubules incubated in the presence of arginine and MSO
AOA
Levels in the particulate fraction (nmol/mg dry weight) Glutamate Ornithine
+
7.7 + 0.3 0.8 _+- 0.1"
6.6 _+ 0.1 12.4 -+ 1.2"
Urea production (nmol/h per mg dry weight)
73.5 + 0.3 93.9 -+ 3.6*
Kidney-cortex tubules were incubated in the presence of 5 mM MSO either without or with 2 mM AOA. After 60 min of incubation, tubules were treated with digitonin and centrifuged as described in Materials and Methods. Significant differences between values measured with AOA against the corresponding ones determined without AOA are noted: *P < 0.01. to be a competitive inhibitor of renal mitochondrial arginase (Fig. 2B), with K, equal to 1.5 -+ 0.1 mM, particularly because the substrate t-arginine carries a positive charge at physiological pH.
Urea Production in Isolated Kidney-Cortex Tubules of Rabbit Under Conditions Differentiating Intracellular Glutamate Accumulation Because kidney cortex mitochondria exhibit omithine aminotransferase (15), omithine derived from arginine via the arginase reaction may react with 2-oxoglutarate, forming glutamate and glutamate-ysemialdehyde. Thus, glutamate may be considered as feedback inhibitor of rabbit kidneycortex arginase. To check whether the inhibitory effect of glutamate on kidney cortex arginase activity may be of physiological importance, we measured urea production in isolated rabbit renal tubules incubated with arginine and methionine sulfoximine (MSO), an inhibitor of glutamine synthetase (32), because M S O is known to elevate glutamate content in rabbit renal tubules (25). As shown in Table 3, glutamate was also accumulated in the particulate fraction isolated from renal tubules after digitonin treatment and centrifugation (41). With the addition of A O A , an inhibitor of aminotransferases (17), a marked decrease of glutamate generation via the reaction catalysed by ornithine aminotransferase resulted in a decline of glutamate level in the particulate fraction containing mitochondria accompanied by an enhancement of urea production for about 30%, despite 2-fold increase of content of ornithine, another inhibitor of arginase (35). In view of observations that levels of 2-oxoglutarate in the particulate fraction of rabbit renal tubules were low and varied between 0.05 and 0.15/amol/g dry weight under various experimental conditions (25) and glutamate concentration in the particulate fraction of rabbit renal tubules could reach 9 / l m o l / g tubule dry weight (25), it is likely that glutamate might inhibit the arginase activity in kidney-cortex tubules under physiological conditions. In summary, the data are consistent with the view that
despite sharing many similar physicochemical features, the hepatic and renal arginases are significantly different with respect to their regulatory properties. Both 2-oxoglutarate and glutamate appear to be inhibitors of rabbit renal arginase with no influence on the liver mitochondrial and cytosolic enzymes. We thank Mr. Tadeusz Lietz for performance of experiments with the use of rabbit renal tubules and Mr. Robert Jarzyna for assistance in the preparation of the manuscript. Technical assistance of Miss B. D~browska is acknowledged. This investigation was supported by the State Committee fi~r Scientific Research (Project 6 PO4A 024 08).
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