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Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes
Comparative Biochemistry and Physiology Part B 119 (1998) 527 – 537
Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes Paula K. Davis, Guoyao Wu * Faculty of Nutrition and Department of Animal Science, Texas A & M Uni6ersity, College Station, Texas 77843 -2471, USA Received 24 June 1997; accepted 3 December 1997
Abstract We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3− and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Urea cycle enzymes; Compartmentation; Kinetics; Citrulline; Arginine; Enterocytes; Pig
1. Introduction The mammalian liver is generally believed to be the only organ in which urea is formed from ammonia via the urea cycle [22]. The enzymes of this cycle include carbamoyl phosphate synthase-I (CPS-I, EC 6.3.4.16), ornithine carbamoyltransferase (OCT, EC 2.1.3.3), argininosuccinate synthase (ASS, EC 6.3.4.5), argininosuccinate lyase (ASL, EC 4.3.2.1), and arginase (EC 3.5.3.1). CPS-I and OCT are exclusively present in the liver and small intestine of mammals, whereas ASS, ASL and arginase are widespread in animal tissues and cells [23,28]. In the liver, the urea cycle spans two * Corresponding author. Tel.: +1 409 8451817; fax: + 1 409 8455292. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00014-5
compartments: mitochondria (CPS-I and OCT) and the cytosol (ASS, ASL and arginase), which functions to efficiently remove ammonia and HCO3− produced from amino acid oxidation [6,7,22]. The view of an important role for the hepatic urea cycle in regulating acid/base balance is the more recent addition to the discussion on the urea cycle function [2]. However, the old and new contention is that the major function of the hepatic urea cycle is to convert ammonia into water-soluble and nontoxic urea [3,17]. We have recently reported that enterocytes of postweaning pigs have all the enzymes required for the synthesis of urea from extracellular and intramitochondrially generated ammonia [33], indicating that the liver is not the only organ capable of ureagenesis in mammals. Despite its relatively low activity, the intestinal
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synthesis of urea from ammonia may be a first line of defence against the potential toxicity of ammonia produced by the extensive intestinal degradation of glutamine and derived from diet and luminal microorganisms. The presence of CPS-I, OCT, ASS and ASL in enterocytes also provides a biochemical basis for synthesizing citrulline and arginine from glutamine by the small intestine [38,39]. The activity of arginase, the final enzyme of the urea cycle, plays a key role in modulating the release by the small intestine of endogenously synthesized and diet-derived arginine into the portal vein [30,37], and therefore, in regulating arginine homeostasis in the body. To the best of our knowledge, there is little or no published information on the compartmentation or kinetics of urea cycle enzymes in the small intestine or enterocytes. A lack of such knowledge has impaired our understanding of arginine metabolism in the gut as well as its role in maintaining arginine homeostasis in mammals. The objective of this study was to determine the compartmentation and kinetics of urea cycle enzymes in enterocytes of the pig, which is widely used as an animal model for studying human intestinal physiology and metabolism [33,38].
2. Materials and methods
2.1. Chemicals L-Amino acids, L-argininosuccinate, D-glucose, ammonium chloride, NaHCO3, bovine serum albumin (BSA, fraction V, essentially fatty-acids free), HEPES, ornithine carbamoyltransferase (from streptococcus faecalis), N-acetylglutamate, ATP, DL-dithiothreitol (DTT), sucrose, EDTA, phenylmethylsulfonyl fluoride, aprotinin, chymostatin, pepstatin, o-phthaldialdehyde (OPA), 3-[N-morpholino]propanesulfonic acid (MOPS), and digitonin were purchased from Sigma (St. Louis, MO). HPLC-grade methanol and water were obtained from Fisher Scientific (Houston, TX).
2.2. Animals and the isolation of jejunum and li6er Pigs were offspring of Yorkshire×Landrace sows and Duroc× Hampshire boars, and were obtained from the Swine Center of Texas A and M University. Piglets were processed (castrated, needle teeth clipped, iron shot and ear notched) by 2 days of age. Piglets were weaned at 21 days of age to a corn and soybeanmeal based diet (20% protein) that met the NRC requirements of all nutrients [35]. At 60 days of age, the jejunum and liver were dissected from anesthetized pigs, as previously described [33]. The experiments were carried out in accordance with the guidelines of the National Research Council for the care and use of
animals, and were approved by Texas A and M University’s Institutional Animal Care and Use Committee.
2.3. Preparation of enterocytes Enterocytes were prepared from the pig jejunum, using oxygenated (95% O2/5% CO2) Ca2 + -free Krebs– Henseleit bicarbonate (KHB) buffer supplemented with 5 mM EDTA, as previously described [33]. Briefly, the jejunum (50 cm) was thoroughly rinsed with 0.9% NaCl to remove its luminal contents. It was then rinsed with 3× 50 ml of oxygenated (95% O2/5% CO2) Ca2 + -free KHB buffer (pH 7.4) containing 20 mM HEPES and 5 mM EDTA. The jejunum was filled with 60 ml of Ca2+free KHB buffer and placed in a flask containing 200 ml of this buffer for incubation at 37oC for 50 min. At the end of the incubation, the jejunum was gently patted with the finger-tips for 1 min, and the lumen was drained into polystyrene tubes. The pellet (enterocytes) was obtained by centrifugation at 400× g for 3 min.
2.4. Preparation of cytosolic and mitochondrial fractions of enterocytes for enzyme assays Enterocytes (40 mg protein) were washed twice with 10 ml of 250 mM sucrose/1 mM EDTA/50 mM potassium phosphate buffer (pH 7.5) containing protease inhibitors (5 mg ml − 1 phenylmethylsulfonyl fluoride, 5 mg ml − 1 aprotinin, 5 mg ml − 1 chymostatin and 5 mg ml − 1 pepstatin), by centrifugation at 400× g and 4oC for 3 min. Cells were homogenized at 4oC in 4 ml of this buffer containing 2.5 mM DTT, and the homogenates were centrifuged at 600×g and 4oC for 15 min. The supernatant was centrifuged at 14000 × g and 4oC for 15 min to obtain the cytosol (supernatant fluid) and mitochondria (pellet). The pellet was washed twice with 2 ml of 250 mM sucrose/1 mM EDTA/50 mM potassium phosphate buffer (pH 7.5) containing 2.5 mM DTT and protease inhibitors (5 mg ml − 1 phenylmethylsulfonyl fluoride, 5 mg ml − 1 aprotinin, 5 mg ml − 1 chymostatin and 5 mg ml − 1 pepstatin), and were then suspended in 1 ml of this buffer. Mitochondria were lysed by three cycles of freezing (in liquid nitrogen) and thawing (at 37oC), and the lysates were centrifuged at 10000×g and 4oC for 15 min. The supernatant was used for enzyme assays.
2.5. Preparation of cytosolic and mitochondrial fractions of enterocytes for measurements of metabolite concentrations Enterocytes were incubated at 37oC for 30 min in the presence of 5 mM glucose, 0.2 mM NH4Cl and plasma concentrations of all amino acids [35]. At the end of the incubation, cells were washed three times with 10 ml of fresh KHB buffer containing 2.5% dialyzed BSA to
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remove extracellular amino acids, which was confirmed by HPLC analysis of amino acids [39]. Cytosolic and mitochondrial fractions were prepared as previously described [40]. Briefly, a 2 ml microcentrifuge tube was filled with 0.2 ml of 1.5 M HClO4 overlaid by a 0.7 ml of inert silicone oil [36]. Enterocyte suspension (0.5 ml) was rapidly mixed with 1.7 ml of an ice-old medium containing 0.25 M sucrose, 20 mM MOPS buffer (pH 7.0), 3 mM EDTA, and 1 mg ml − 1 digitonin. After 40 s, 0.5 ml of the cell mixture was loaded onto the top layer of the microcentrifuge tube for centrifugation (3000×g, 0oC, 20 s). The top layer (cytosolic fraction) and the bottom acid layer (mitochondria) were used for analysis of ammonia, amino acids, carbamoyl phosphate, argininosuccinate and ATP, as previously described [33 – 39]. Substrate concentrations in the cytosol and mitochondria were calculated on the basis of water volume in enterocytes (437958 nl per 106 cells, mean9SEM, n = 10) and mitochondrial matrix volume (1.219 0.28 ml mg − 1 protein, mean9 SEM, n = 10), respectively. Water volume in enterocytes and mitochondrial matrix volume were determined by incubating cells or mitochondria in the presence of 3H2O and [14C]sucrose, as previously described [15,36].
2.6. Measurements of the acti6ities of urea cycle enzymes in the cytosol and mitochondrial fractions of enterocytes The activities of CPS-1, OCT, ASS and ASL were determined at 37oC as previously described [33]. All the enzyme assays were performed at two protein levels and three time points (0, 10 and 20 min). Briefly, the assay mixture for CPS-1 (0.5 ml) consisted of 0.15 M potassium phosphate buffer (pH 7.5), 25 mM ATP, 25 mM MgCl2, 5 mM N-acetylglutamate, 20 mM NH4Cl, 5 mM ornithine, 100 mM NaHCO3, 10 U of added ornithine carbamoyltransferase, and mitochondrial extracts (0.5 mg protein). The reaction mixtures were prepared in 0.15 M potassium phosphate buffer (pH 7.5) and were carefully adjusted to pH 7.5 before starting enzyme reaction. Our CPS-I assay was based on the method developed by Hager and Jones [11] and used by other investigators [18]. In our preliminary studies, we found that the use of phosphate buffer (0.15 M, pH 7.5) decreased CPS-I activity by 18% (n= 6) compared with Tris buffer (0.1 M, pH 7.5). We did not use Tris buffer in CPS-I assay because this buffer contained large amounts of an unknown compound that was eluted closely to the citrulline peak on our HPLC chromatograms, which could result in difficulties in accurate peak integrations. In aqueous solution, free ammonia (NH3) is at equilibrium with ammonium ion (NH4+ ) [8]. At pH 7.5, 37oC, and pKa of 9.2, the ratio of [NH3]/[NH4+ ]
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is 0.02, according the Henderson–Hasselbalch equation: pH=pKa + log ([NH3]/[NH4+ ]) [8]. NH3 and Mg-ATP (rather than NH4+ and ATP) are true substrates for CPS-I. The assay medium for OCT (2.0 ml) contained 0.1 M potassium phosphate buffer (pH 7.5), 15 mM ornithine, 40 mM carbamoyl phosphate, and mitochondrial extracts (0.04 mg protein). Our OCT assay was based on the method of Ceriotti [5]. The ASS assay mixture (0.2 ml) consisted of 75 mM potassium phosphate buffer (pH 7.5), 10 mM citrulline, 5 mM aspartate, 5 mM ATP, 5 mM MgSO4, and cytosolic extracts (0.4 mg protein). Mg-ATP (rather than ATP) is a true substrate for ASS. The ASL assay mixture (40 ml) contained 129 mM sodium phosphate buffer (pH 7.0), 10 mM argininosuccinate, 65 mM EDTA and cytosolic extracts (0.1 mg protein). For arginase assay, a mixture of 100 ml of 10 mM MnCl2 (in 50 mM Tris–HCl buffer, pH 7.5) and 100 ml of mitochondrial or cytosolic extracts (0.5 mg protein) was pre-heated at 55oC for 5 min. After cooling to room temperature, the mixture was incubated for 0, 10 or 20 min with 100 ml of 60 mM arginine (in 50 mM Tris–HCl buffer, pH 7.5). Arginine solution was adjusted to pH 7.5 with 1 N HCl before addition to the assay mixture. Arginase activity was also measured without pre-heating enzyme protein at 55oC before addition of arginine. For all above enzyme assays, the incubation was terminated by addition of 100 ml of 1.5 mM HClO4, and the acidified medium was neutralized with 50 ml of 2 mM K2CO3. The neutralized extracts were used for analysis of citrulline (for CPS-1 and OCT assays), urea and ornithine (for arginase assay), arginine (for ASL assay), and arginine plus argininosuccinate (for ASS assay) as previously described [33]. To assess mitochondrial contamination of the cytosolic fraction, the activity of ornithine aminotransferase (OAT) (a mitochondrial enzyme) in the cytosolic and mitochondrial fractions of enterocytes was determined as previously described [37]. To assess cytosolic contamination of the mitochondrial fraction, the activity of P5C reductase (a cytosolic enzyme in pig enterocytes) in the cytosolic and mitochondrial fractions was also determined as previously described [37]. To assess the capacity for carbamoyl phosphate synthesis in the cytosol by CPS-II (a glutamine-utilizing enzyme) compared with that in mitochondria by CPS-I (an ammonia- utilizing enzyme), CPS-II activity was measured in the cytosol of pig enterocytes as described for CPS-I, except that 20 mM L-glutamine replaced 20 mM NH4Cl in the assay mixture [11]. Protein in the cytosol and mitochondria was determined by a modified Lowry method using BSA as standard [20]. Enzyme activities were expressed on the basis of protein.
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Fig. 1. Activities of carbamoylphosphate synthase-I (CPS-I) at various substrate concentrations in mitochondria of porcine enterocytes. Data are mean9 SEM, n = 5. (A) Varying [NH4+ ] at constant [ATP] (30 mM) and [HCO3− ] (100 mM); increasing [NH4+ ] from 0.5 to 20 mM increased (P B0.05) CPS-I activity. (B) Varying [HCO3− ] at constant [ATP] (30 mM) and [NH4+ ] (20 mM); increasing [HCO3− ] from 5 to 100 mM increased (P B0.05) CPS-I activity. (C) Varying [ATP] at constant [NH4+ ] (20 mM) and [HCO3− ] (100 mM); increasing [ATP] from 1 to 30 mM increased CPS-I activity. CP, carbamoylphosphate.
2.7. Determination of Vmax and Km 6alues for enterocyte urea cycle enzymes The assay of CPS-1 was carried out as described above, except: (1) Varying [HCO3− ] from 5 to 100 mM at constant [ATP] (30 mM) and [NH4+ ] (20 mM); (2) varying NH4+ from 0.5 to 25 mM at constant [ATP] (30 mM) and [HCO3− ] (100 mM); (3) varying [ATP] from 1 to 35 mM at constant [HCO3− ] (100 mM) and [NH + ] (20 mM). The assay of OCT was carried out as described above, except: (1) Varying [ornithine] from 1 to 60 mM at constant [CP] (40 mM); (2) varying [CP] from 1 to 60 mM at constant [ornithine] (15 mM). The assay of ASS was carried out as described above, except: (1) varying [citrulline] from 0.02 to 10 mM at constant [ATP] (5 mM) and [aspartate] (5 mM); (2) varying [aspartate] from 0.025 to 5 mM at constant [ATP] (5 mM) and [citrulline] (10 mM); (3) varying
[ATP] from 0.5 to 25 mM at constant [citrulline] (10 mM) and [aspartate] (5 mM). The assays of ASL and arginase were carried out as described above, except that [argininosuccinate] varied from 0.031 to 10 mM and [arginine] varied from 1 to 30 mM, respectively. Vmax and Km values of enzymes were determined according to the plots of s versus 6 in Figs. 1–5, on the basis of the definition that Vmax is the maximum velocity of reaction and Km is the substrate concentration at which enzyme velocity is half-maximal [9].
2.8. Determination of kinetics of urea cycle enzymes in pig li6er To compare kinetics of urea cycle enzymes between porcine enterocytes and liver, the cytosol and mitochondrial fractions of pig livers were prepared as described above for enterocytes. Vmax and Km values of
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Fig. 2. Activities of ornithine carbamoyltransferase (OCT) at various substrate concentrations in mitochondria of porcine enterocytes. Data are mean9 SEM, n = 5. (A) [Ornithine] varied at constant [CP] (40 mM). Increasing [ornithine] from 1 to 12.5 mM increased OCT activity. (B) [Carbamoylphosphate] varied at constant [ornithine] (15 mM). Increasing [carbamoylphosphate] from 1 to 40 mM increased (P B0.05) OCT activity. Cit, citrulline.
CPS-I and OCT in mitochondria and of ASS, ASL and arginase in the cytosol were determined as described above, except that the amount of cytosolic protein used for arginase assay was 5 mg.
2.9. Statistical analysis Data were analyzed by unpaired t-test for comparison of the cytosol and mitochondrial enzyme activities, or by one-way ANOVA with the Student – Newman– Keuls test for comparison of activities of a given enzyme at various substrate concentrations [27]. Probability values less than 0.05 (P B0.05) were taken to indicate statistical significance.
3. Results
3.1. Acti6ities of urea cycle enzymes in the cytosol and mitochondria of enterocytes
3.2. Acti6ities of OAT, P5C reductase and CPS-II in the cytosol and mitochondria of enterocytes Ornithine aminotransferase activity was found only in the mitochondrial fraction of enterocytes, and was not detected in the cytosolic fraction. The activity of P5C reductase was found only in the cytosolic fraction of enterocytes, and was not detected in the mitochondrial fraction. CPS-II activity in the cytosol of pig enterocytes was 0.2939 0.034 nmol min − 1 per mg protein (mean9 SEM, n= 6), which was much lower than CPS-I activity in pig enterocytes (Table 1).
3.3. Kinetics of urea cycle enzymes in enterocytes Changes in activities of urea cycle enzymes with increasing substrate concentrations are illustrated in Figs. 1–5. Vmax and Km values of urea cycle enzymes are summarized in Table 2.
3.4. Carbamoylphosphate synthase-I These data are summarized in Table 1. In porcine enterocytes, CPS-I and OCT were exclusively located in mitochondria, ASS and ASL were found only in the cytosol, and arginase was present in both the cytosol and mitochondria. When enzyme protein was preheated at 55oC before addition of arginine substrate, arginase activity was 3-fold greater in the cytosol than in mitochondria. However, when arginase assay did not involve pre-heating of enzyme protein at 55oC before addition of arginine, arginase activity was found to be similar between the cytosolic and mitochondrial fractions. Thus, preheating of enzyme protein at 55oC had no effect (P\ 0.05) on cytosolic arginase activity, but markedly decreased (P B0.05) mitochondrial arginase activity.
At constant [ATP] (30 mM) and [HCO3− ] (100 mM), increasing [NH4+ ] from 0.5 to 15 mM increased (PB 0.05) CPS-I activity in a concentration-dependent manner, but there was no further increase in the enzyme activity with increasing [NH4+ ] from 15 to 25 mM (Fig. 1A). At constant [NH4+ ] (20 mM) and [ATP] (30 mM), increasing [HCO3− ] from 5 to 100 mM progressively increased (P B0.05) CPS-I activity, but there was no difference (P\ 0.05) in the enzyme activity between 100 and 140 mM [HCO3− ] (Fig. 1B). At constant [NH4+ ] (20 mM) and [HCO3− ] (100 mM), increasing [ATP] from 1 to 30 mM gradually increased (PB 0.05) CPS-I activity, but there was no further increase (P\ 0.05) in the enzyme activity between 30 and 35 mM [ATP] (Fig. 1C).
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Fig. 3. Activities of argininosuccinate synthase (ASS) at various substrate concentrations in the cytosol of porcine enterocytes. Data are mean9 SEM, n =5. (A) [ATP] varied at constant [citrulline] (10 mM) and [aspartate] (5 mM). Increasing [ATP] from 0.5 to 5 mM increased (PB 0.05) ASS activity. (B) [Aspartate] varied at constant [ATP] (5 mM) and [citrulline] (10 mM). Increasing [aspartate] from 0.025 to 0.25 mM increased (P B0.05) ASS activity. (C) [Citrulline] varied at constant [ATP] (5 mM) and [aspartate] (5 mM). Increasing [citrulline] from 0.02 to 1 mM increased ASS activity. AS, argininosuccinate.
3.5. Ornithine carbamoyltransferase At constant [CP] (40 mM), increasing [ornithine] from 1 to 12.5 mM progressively increased (P B 0.05) OCT activity, but there was no further increase in the enzyme activity with increasing [ornithine] from 12.5 to 15 mM (Fig. 2A). Interestingly, increasing [ornithine] from 15 to 20, 30, 40 and 60 mM at constant [CP] (40 mM) progressively decreased (P B0.05) OCT activity by 45.3, 52.4, 57.7 and 63.5% (n =5), respectively. At constant [ornithine] (15 mM), increasing [CP] from 1 to 40 mM gradually increased (P B0.05) OCT activity, but there was no further increase in the enzyme activity between 40 and 60 mM [CP] (Fig. 2B).
3.6. Argininosuccinate synthase At constant [citrulline] (10 mM) and [aspartate] (5 mM), increasing [ATP] from 0.5 to 5 mM increased (P B 0.05) ASS activity in a concentration-dependent manner, but there was no further increase in the en-
zyme activity between 5 and 6 mM [ATP] (Fig. 3A). Interestingly, increasing [ATP] from 6 to 10, 15 and 25 mM at constant [citrulline] (10 mM) and [aspartate] (5 mM) progressively decreased (P B 0.05) ASS activity by 70.4, 92.5 and 96.0%, respectively. At constant [ATP] (5 mM) and [citrulline] (10 mM), increasing [aspartate] from 0.025 to 0.25 mM increased (PB 0.05) ASS activity in a concentration-dependent manner, but there was no further increase in the enzyme activity between 0.25 and 5 mM aspartate (Fig. 3B). At constant [ATP] (5 mM) and [aspartate] (5 mM), increasing [citrulline] from 0.02 to 1 mM increased (PB 0.05) ASS activity, but there was no difference in the enzyme activity between 1 and 10 mM citrulline (Fig. 3C).
3.7. Argininosuccinate lyase Increasing [argininosuccinate] from 0.031 to 2.5 mM progressively increased (PB0.05) ASL activity in a concentration-dependent manner. There was no difference (P\0.05) in ASL activity between 2.5 and 10 mM argininosuccinate (Fig. 4).
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Table 1 The activities of urea cycle enzymes and ornithine aminotransferasea
Fig. 4. The activities of argininosuccinate lyase (ASL) at various substrate concentrations in the cytosol of porcine enterocytes. Data are mean9 SEM, n = 5. Increasing [argininosuccinate] from 0.031 to 2.5 mM increased (P B0.05) ASL activity. There was no difference (P \0.05) in ASL activity between 2.5 and 10 mM argininosuccinate. Arg, arginine.
3.8. Arginase When arginase assay involved preheating enzyme protein at 55oC before addition of arginine, increasing [arginine] from 1 to 20 mM progressively increased (P B 0.05) cytosolic and mitochondrial arginase activity in a concentration-dependent manner, but there was no difference (P\ 0.05) in the enzyme activity between 20 and 30 mM arginine (Fig. 5A). When enzyme protein was not preheated at 55oC, increasing [arginine] from 1 to 20 mM also progressively increased (P B 0.05) cytosolic and mitochondrial arginase activity in a concentration-dependent manner, and there was no difference (P\ 0.05) in the enzyme activity between the cytosolic and mitochondrial fractions at each arginine concentration used (Fig. 5B). Preheating enzyme protein at 55oC decreased (PB0.05) Vmax value of mitochondrial
Enzyme
Cytosol (nmol min−1 per mg protein)
Mitochondria
CPS-I OCT ASS ASL Arginase (preheated at 55oC) Arginase (no preheating) OAT P5C reductase
ND " ND " 1.91 90.13 3.57 90.24 7.32 9 0.39
6.52 9 0.25 708.2 9 46.7 ND " ND " 2.01 90.17*
7.16 9 0.54
7.28 9 0.65
ND§ 63.7 9 8.2
362.4 9 12.8 ND§
Enzyme activities were measured as described in Section 2. Data are mean 9SEM, n = 6. CPS-1, carbamoylphosphate synthase-1; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; OAT, ornithine aminotransferase; P5C, pyrroline-5-carboxylate; ND, not detected. " Detection limit was 100 pmol ml−1 of sample; § Detection limit was 7 nmol ml−1 of sample. a In the cytosol and mitochondria of porcine enterocytes. * PB0.01: different from the cytosol, as analyzed by unpaired t-test.
arginase but had no effect (P\ 0.05) on the cytosolic enzyme (Table 2). Km values were similar between these two isoforms of arginase (Table 2).
3.9. Concentrations of urea cycle enzyme substrates in pig enterocytes These data are summarized in Table 3. The cytosol of enterocytes contained relatively large amounts of ATP, aspartate and carbamoylphosphate, and to a lesser extent, ammonia, ornithine, citrulline and arginine. There was a small amount of argininosuccinate in the cytosol of enterocytes. Mitochondria contained rela-
Fig. 5. The activities of cytosolic and mitochondrial arginase at various substrate concentrations in porcine enterocytes. Data are mean 9SEM, n=5. (A) Arginase activities were measured by pre-heating enzyme protein at 55°C before addition of arginine substrate. Increasing [arginine] from 1 to 20 mM increased (PB 0.05) arginase activity. (B) Arginase activities were measured without pre-heating enzyme protein before the addition of arginine substrate. Increasing [arginine] from 1 to 20 mM increased (P B0.05) arginase activity.
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Table 2 Vmax and Km of urea cycle enzymes in the cytosol or mitochondria of porcine enterocytes and liver Enzyme
Substrate
Enterocytes Vmax
Km
Vmax
Km
6.409 0.37 6.889 0.43 6.759 0.26
1.34 90.11 58.6 9 2.4 15.2 9 1.4
18.6 90.23* 19.0 9 0.16* 18.3 9 0.21*
1.20 9 0.16 5.77 9 0.49* 1.69 9 0.14*
713.4 9 22.3 699.59 32.1
5.13 90.24 17.1 91.2
915.7 9 68.2* 926.3 9 73.0*
1.58 9 0.12* 0.46 9 0.05*
Mg-ATP Asp Cit
1.849 0.10 1.939 0.07 1.909 0.08
2.86 90.16 0.054 9 0.003 0.15 9 0.01
8.58 90.72* 8.36 90.53* 8.42 90.51*
0.24 9 0.03* 0.031 90.002* 0.068 90.01*
CPS-I (Mit)
NH+ 4 HCO− 3 Mg-ATP
OCT (Mit)
Orn CP
ASS (Cyt)
AS
3.509 0.23
0.63 9 0.04
17.7 90.19*
0.12 9 0.01*
a
Arg
7.13 90.60
7.46 9 0.52
3072 9 256*
3.38 9 0.26*
a
Arg
1.9390.14
7.09 90.58
ASL (Cyt) Arginase (Cyt)
Liver
Arginase (Mit)
—
—
Data are mean 9 SEM in nmol min−1 per mg protein for Vmax and mM for Km; n = 5. CPS-1, carbamoylphosphate synthase-1; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; CP, carbamoyl phosphate; AS, argininosuccinate; Cyt, cytosol; Mit, mitochondria. a Enzyme protein was preheated at 55°C before addition of arginine. * PB0.01: Different from the corresponding value for enterocytes, as analyzed by unpaired t-test.
tively large amounts of ammonia, ATP, aspartate, and to a lesser extent, ornithine, citrulline, arginine and carbamoylphosphate.
3.10. Kinetics of urea cycle enzymes in the li6er Vmax and Km values of CPS-I, OCT, ASS, ASL and arginase are summarized in Table 2. In the liver, arginase had the greatest activity among the urea cycle enzymes, followed by OCT, CPS-I, ASL and ASS. Except for OCT, Vmax values of urea cycles enzymes were much greater (P B 0.01) in the porcine liver than in enterocytes, and vice versa for Km values of these enzymes.
Table 3 Concentrations of urea cycle enzyme substrates in pig enterocytes Substrate
Cytosol
Mitochondria
Ammonia ATP Aspartate Citrulline Arginine Ornithine Argininosuccinate Carbamoylphosphate
0.459 0.06 4.289 0.51 5.569 0.62 0.3690.04 0.8490.09 0.309 0.03 0.0429 0.005 2.26 9 0.19
1.189 0.07* 8.56 9 0.93* 2.419 0.33* 0.529 0.06* 0.759 0.08 0.619 0.07* ND 0.599 0.07*
Data are mean 9 SEM in mM; n= 6. ND, not detectable. Detection limit for amino acid analysis by our HPLC method was 100 pmol ml−1 of sample. * PB0.01: Different from the cytosol value, as analyzed by unpaired t-test.
4. Discussion The results of this study demonstrate that in porcine enterocytes: (1) CPS-I and OCT were exclusively located in mitochondria; (2) ASS and ASL were found only in the cytosol; and (3) arginase was present in both the cytosol and mitochondria (Table 1). To the best of our knowledge, this is the first report of compartmentation of urea cycle enzymes in enterocytes. As in the liver [22], intestinal urea synthesis from ammonia spans two compartments: the cytosol and mitochondria. Among urea cycle enzymes in enterocytes, the activity of OCT was the highest, followed by arginase, CPS-I, ASL and ASS in decreasing order. The cytosolic arginase appears to differ from the mitochondrial enzyme in that the latter, but not the former, is inactivated by pre-heating of enzyme protein at 55oC. Interestingly, when enzyme protein was not preheated at 55oC, arginase activity was equally distributed between the cytosol and mitochondria. Thus, two isoforms of arginase are present in porcine enterocytes, as reported in the rat small intestine on the basis of electrophoretic separation [12]. This is consistent with the recent report that porcine enterocytes contain anionic and non-anionic isoforms of arginase and that preheating enzyme protein (60oC, 15 min) decreased arginase activity in suckling pigs enterocytes [24]. In the study of M’Rabet-Touil et al. [24], it was not known whether cytosolic or mitochondrial arginase in porcine enterocytes was sensitive to heat inactivation because porcine enterocytes were stored at − 80oC and homogenized by sonification, thereby releasing both cytosolic and mitochondrial enzymes into the same supernatant fluid used for the arginase assay.
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To the best of our knowledge, this is the first report of the kinetics of all urea cycle enzymes in enterocytes. Km values of ASS for ATP, aspartate and citrulline were much lower than the concentrations of these substrates in the cytosol of enterocytes, suggesting a relatively high affinity of ASS for its substrates. However, Vmax value of ASS was the lowest among all urea cycle enzymes in enterocytes. Carbamoylphosphate concentrations in mitochondria of enterocytes were much lower than the Km value of CPS-I for this substrate, which may explain a relatively low activity of CPS-I in pig enterocytes. These results suggest that ASS and carbamoylphosphate may be rate-limiting factors in the synthesis of citrulline from glutamine in enterocyte mitochondria. Similarly, Km values of OCT, ASL and arginase for ornithine, argininosuccinate and arginine (Table 2) were much greater than concentrations of these substrates in the cytosol or mitochondria of enterocytes (Table 3), suggesting that rates of fluxes through these enzymes are far from maximal in intact enterocytes. The relatively high Km value of OCT for carbamoylphosphate compared to its intramitochondrial concentrations may explain preferential channelling of extracellular arginine or ornithine into OAT for the synthesis of P5C and therefore of proline in pig enterocytes [37]. Although porcine intestinal OCT activity was inhibited by excess amounts of ornithine (\ 15 mM), as reported for purified porcine liver OCT [16], this inhibition may not be of physiological relevance because a much greater concentration of ornithine is required for enzyme inhibition than ornithine concentration in enterocyte mitochondria (0.81 mM) (Table 3). Substrate inhibition of enzymes was also observed for porcine intestinal ASS when [ATP] was greater than 6 mM. This inhibition is not likely to be physiologically relevant on the basis of ATP concentrations in the cytosol of enterocytes (4.28 mM) (Table 3). Kinetics of hepatic urea cycle enzymes appears to be similar between pigs (Table 2) and rats [22]. However, in pigs, kinetics of urea cycle enzymes differs remarkably between enterocytes and the liver. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values (Table 2). Urea cycle enzymes also differ between porcine enterocytes and liver in their responses to physiological and nutritional alterations. For example, starvation, feeding a high protein diet and glucagon treatment increase both the activities and mRNA levels of all urea cycle enzymes including OCT in the liver [23,28]. In contrast, glucagon treatment has been shown to have no effect on the activities and mRNA levels of intestinal CPS-I or OCT [25]. Also, feeding a high protein diet has been reported to decrease the activity and mRNA level of intestinal OCT [31,32]. The difference in enzyme expressions between the liver and small intestine in response to protein
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feeding is not restricted to urea cycle enzymes, as feeding a high protein diet increases OAT activity in the liver but decreases the enzyme activity in the small intestine [21]. The enzymological differences between the liver and small intestine in response to physiological and nutritional alterations may be related to the major metabolic functions of these two organs. For example, an increase in dietary protein intake necessitates an increase in the activities of hepatic urea cycle enzymes to remove ammonia produced from amino acid oxidation. On the other hand, in mammals fed a high protein diet, there is no need for increasing intestinal production of citrulline for endogenous synthesis of arginine. The urea cycle enzymes in postweaning pig enterocytes may have dual functions: (1) Synthesizing citrulline from ammonia, HCO3− and ornithine; and (2) converting ammonia, HCO3− and aspartate into urea. Both cytosolic and mitochondrial isoforms of arginase are induced in enterocytes of postweaning pigs (Table 1) [33]. As a result, enterocytes shift from a net synthesis of arginine in neonates [10,38] to a net release of citrulline in postweaning pigs [39]. Because of a low rate of urea synthesis from ammonia in enterocytes compared with the liver [22], we suggest that the presence of enterocyte CPS-I and OCT may function primarily to synthesize citrulline from ammonia, HCO3− and ornithine in postweaning pigs. This suggestion is consistent with the view of Campbell [6] regarding the function of intestinal CPS-I and OCT in ureotelic species. The co-localization of CPS-I and OCT and a high activity of OCT in enterocyte mitochondria favors the synthesis of citrulline from ammonia, HCO3− and ornithine in the pig small intestine, as in the mammalian liver [26]. The location of OAT in mitochondria greatly facilitates the provision of ornithine from glutamine in enterocytes. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes which have a relatively high activity of arginase. Such kinetic properties of intestinal urea cycle enzymes help to maximize the net synthesis of citrulline from glutamine [39], and explain the release of large amounts of citrulline from the small intestine of postweaning pigs [35]. Ornithine aminotransferase and P5C reductase are required for proline synthesis from glutamine and arginine. Glutamine is metabolized to P5C via phosphate-dependent glutaminase and P5C synthase in enterocyte mitochondria [38]. Some of the glutamine-derived P5C is then transported from mitochondria into the cytosol for proline synthesis by P5C reductase. The co-localization of arginase and OAT in enterocyte mitochondria also facilitates the synthesis of P5C from arginine, because the arginine-derived ornithine is efficiently channelled to OAT. Argininederived P5C is then transported from mitochondria
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Fig. 6. Compartmentation of the synthesis of proline from glutamine and arginine in enterocytes. PDG, phosphate-dependent glutaminase; P5C, pyrroline-5-carboxylate; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; NOS, nitric oxide synthase; BH4, tetrahydrobiopterin; NO, nitric oxide.
into the cytosol for proline synthesis. This may help to explain, in part, the much speculated role of mitochondrial arginase in extrahepatic tissues and cells [13]. These compartmentalized reactions for proline synthesis are summarized in Fig. 6. The role of OAT in proline synthesis is substantiated by our finding that an inhibition of this enzyme leads to a decrease in proline synthesis from arginine by 80 – 85% in pig enterocytes [37]. It is worth pointing out that the location of P5C reductase in the cytosol of enterocytes prevents or minimizes a potential recycling of glutamine-derived P5C into proline, thereby ensuring net synthesis of citrulline from glutamine. By regulating the hydrolysis of arginine into ornithine (an inhibitor of P5C synthase [19]), mitochondrial arginase may play an important role in regulating the intestinal synthesis of P5C and therefore of citrulline and proline from glutamine. On the other hand, cytosolic arginase may play a critical role in modulating intracellular concentrations of arginine for other competing arginine-requiring pathways such as NO synthesis (Fig. 6) as in macrophages [29] and endothelial cells [4]. An alteration of NO production may, in turn, contribute to the regulation of intestinal function [1]. Cytosolic arginase may also regulate the provision of
ornithine for ornithine decarboxylase (a cytosolic enzyme) (Fig. 6), the first and key regulatory enzyme in the synthesis of polyamines, thereby regulating the proliferation and differentiation of intestinal epithelial cells [14]. In conclusion, the urea cycle enzymes in porcine enterocytes span two compartments: mitochondria (CPS-I, OCT and arginase) and the cytosol (ASS, ASL and arginase). Except for OCT, Vmax values of urea cycle enzymes are much lower in enterocytes than in the liver, and vice versa for their Km values. The compartmentation and kinetics properties of urea cycle enzymes provide a biochemical basis for explaining net synthesis of large amounts of citrulline from glutamine in enterocytes of postweaning pigs. The presence of arginase in mitochondria and the cytosol may play an important role in regulating the metabolism of proline, nitric oxide and polyamines in enterocytes.
Acknowledgements We thank Wene Yan, Nick E. Flynn, Sean P. Flynn and Darrell A. Knabe for technical assistance, as well as Pat Onstott and Frances Mutscher for secretarial
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support. This research was supported by USDA competitive grants c 94-37206-1100 and c 97-35206-5096 (to G. Wu).
References [1] Alican I, Kubes P. A critical role for nitric oxide in intestinal barrier function and dysfunction. Am J Physiol 1996;270:G225– 37. [2] Atkinson DE, Bourke E. The role of ureagenesis in pH homeostasis. Trends Biochem Sci 1984;9:297–300. [3] Boon L, Blommaart PJE, Meijer AJ, Lamers WH, Schoolwerth AC. Response of hepatic amino acid consumption to chronic metabolic acidosis. Am J Physiol 1996;271:198–202. [4] Buga GM, Singh R, Pervin S, Rogers NE, Schmitz DA, Jenkinson CP, Cederbaum SD, Ignarro LJ. Arginase activity in endothelial cells: inhibition by N-hydroxy-L-arginine during high-output NO production. Am J Physiol 1996;271:1988– 98. [5] Ceriotti G. Ornithine carbamoyltransferase. In: Bergmeyer HU, editor. Methods of Enzymatic Analysis. Weinheim: Verlag Chemie, 1974:691 – 8. [6] Campbell JW. Mitochondrial ammonia metabolism and the proton-neutral theory of hepatic ammonia detoxication. J Exp Zool 1997;278:308 – 21. [7] Cohen NS, Kuda A. Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: An immunocytochemical study. J Cell Biochem 1996;60:334–40. [8] Cooper AJL. Ammonia metabolism in mammals. Adv Exp Med Biol 1994;341:21 – 37. [9] Cornish-Bowden A. Fundamentals of Enzyme Kinetics. Revised Edition. London: Portland Press, 1995. [10] Flynn NE, Wu G. An important role for endogenous synthesis of arginine in maintaining arginine homeostasis in neonatal pigs. Am J Physiol 1996;271:1149–55. [11] Hager SE, Jones ME. Initial steps in pyrimidine synthesis in Ehrlich ascites carcinoma in vitro. J Biol Chem 1967;242:5667– 73. [12] Herzfeld A, Raper SM. Enzymes of ornithine metabolism in adult and developing rat intestine. Biochim Biophys Acta 1976;428:600 – 10. [13] Jenkinson CP, Grody WW, Cederbaum SD. Comparative properties of arginase. Comp Biochem Physiol 1996;114B:107– 32. [14] Johnson LR. Regulation of gastrointestinal mucosal growth. Physiol Rev 1988;68:456–502. [15] Jois M, Ewart HS, Brosnan JT. Regulation of glycine catabolism in rat liver mitochondria. Biochem J 1992;283:435–9. [16] Koger JB, Howell RG, Kelly M, Jones EE. Purification and properties of porcine liver ornithine transcarbamylase. Arch Biochem Biophys 1994;309:293–9. [17] Krebs HA. The discovery of the ornithine cycle. In: Grisolia S, Baguena R, Mayor F, editors. The Urea Cycle. New York: Wiley, 1976:1 – 12. [18] Levine RL, Kretchmer N. Conversion of carbamoylphosphate to hydroxyurea. An assay for carbamoylphosphate synthetase. Anal Biochem 1971;42:324–37. [19] Lodato RF, Smith RJ, Valle D, Phang JM, Aoki TT. Regulation of proline biosynthesis: The inhibition of pyrroline-5-carboxylate synthase activity by ornithine. Metabolism 1981;30:908– 13.
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[20] Markwell MAK, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simply protein determination in membrane and lipoprotein samples. Anal Biochem 1978;87:206– 12. [21] Matsuzawa T, Kobayashi T, Tashiro K, Kasahara M. Changes in ornithine metabolic enzymes induced by dietary protein in small intestine and liver: Intestine-liver relationship in ornithine supply to liver. J Biochem 1994;116:721 – 7. [22] Meijer AJ, Lamers WH, Chamuleau AFM. Nitrogen metabolism and ornithine cycle function. Physiol Rev 1990;70:701 –48. [23] Morris SM Jr. Regulation of enzymes of urea and arginine synthesis. Annu Rev Nutr 1992;12:81 – 101. [24] M’Rabet-Touil H, Lerminiaux H, Duee PH, Blachier F. Evidence for increased anionic arginase activity in pig enterocytes during development. Biochem Mol Biol Int 1996;38:197–204. [25] Ryall JC, Quantz MA, Shore GC. Rat liver and intestinal mucosa differ in the developmental pattern and hormonal regulation of carbamoylphosphate synthetase I and ornithine carbamoyltransferase gene expression. Eur J Biochem 1986;156:453 – 8. [26] Srere PA. Complexes of sequential metabolic enzymes. Annu Rev Biochem 1987;56:89 – 124. [27] Steel RGD, Torrie JH. Principles and Procedures of Statistics. New York: McGraw-Hill, 1980. [28] Takiguchi M, Mori M. Transcriptional regulation of genes for ornithine cycle enzymes. Biochem J 1995;312:649 – 59. [29] Wang WW, Jenkinson CP, Griscavage JM, Kern RM, Arabolos NS, Byrns RE, Cederbaum SD, Ignarro LJ. Co-induction of arginase and nitric oxide synthase in murine macrophages activated by lipopolysaccharide. Biochem Biophys Res Commun 1995;210:1009– 16. [30] Windmueller HG, Spaeth AE. Metabolism of absorbed aspartate, asparagine, and arginine by rat small intestine in vivo. Arch Biochem Biophys 1976;175:670 – 6. [31] Wraight C, Hoogenraad N. Dietary regulation of ornithine transcarbamylase mRNA in liver and small intestine. Aust J Biol Sci 1985;41:435 – 42. [32] Wraight C, Lingelbach K, Hoogenraad N. Comparison of ornithine transcarbamylase from rat liver and intestine. Evidence for differential regulation of enzyme levels. Eur J Biochem 1985;153:239 – 42. [33] Wu G. Urea synthesis in enterocytes of developing pigs. Biochem J 1995;312:717 – 23. [34] Wu G. An important role for pentose cycle in the synthesis of citrulline and proline from glutamine in porcine enterocytes. Arch Biochem Biophys 1996;336:224 – 30. [35] Wu G, Borbolla AG, Knabe DA. The uptake of glutamine and release of arginine, citrulline and proline by the small intestine of developing pigs. J Nutr 1994;124:2437 – 44. [36] Wu G, Flynn NE. Regulation of glutamine and glucose metabolism by cell volume in lymphocytes and macrophages. Biochim Biophys Acta 1995;1243:343– 50. [37] Wu G, Knabe DA, Flynn NE, Yan W, Flynn SP. Arginine degradation in developing porcine enterocytes. Am J Physiol 1996;271:G913– 9. [38] Wu G, Knabe DA. Arginine synthesis in enterocytes of neonatal pigs. Am J Physiol 1995;269:R621– 9. [39] Wu G, Knabe DA, Flynn NE. Synthesis of citrulline from glutamine in pig enterocytes. Biochem J 1994;299:115 –21. [40] Zuurendonk PF, Tager JM. Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells. Biochim Biophys Acta 1974;333:393 – 9.
Compartmentation and kinetics of urea cycle enzymes in porcine enterocytes Paula K. Davis, Guoyao Wu * Faculty of Nutrition and Department of Animal Science, Texas A & M Uni6ersity, College Station, Texas 77843 -2471, USA Received 24 June 1997; accepted 3 December 1997
Abstract We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3− and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Urea cycle enzymes; Compartmentation; Kinetics; Citrulline; Arginine; Enterocytes; Pig
1. Introduction The mammalian liver is generally believed to be the only organ in which urea is formed from ammonia via the urea cycle [22]. The enzymes of this cycle include carbamoyl phosphate synthase-I (CPS-I, EC 6.3.4.16), ornithine carbamoyltransferase (OCT, EC 2.1.3.3), argininosuccinate synthase (ASS, EC 6.3.4.5), argininosuccinate lyase (ASL, EC 4.3.2.1), and arginase (EC 3.5.3.1). CPS-I and OCT are exclusively present in the liver and small intestine of mammals, whereas ASS, ASL and arginase are widespread in animal tissues and cells [23,28]. In the liver, the urea cycle spans two * Corresponding author. Tel.: +1 409 8451817; fax: + 1 409 8455292. 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)00014-5
compartments: mitochondria (CPS-I and OCT) and the cytosol (ASS, ASL and arginase), which functions to efficiently remove ammonia and HCO3− produced from amino acid oxidation [6,7,22]. The view of an important role for the hepatic urea cycle in regulating acid/base balance is the more recent addition to the discussion on the urea cycle function [2]. However, the old and new contention is that the major function of the hepatic urea cycle is to convert ammonia into water-soluble and nontoxic urea [3,17]. We have recently reported that enterocytes of postweaning pigs have all the enzymes required for the synthesis of urea from extracellular and intramitochondrially generated ammonia [33], indicating that the liver is not the only organ capable of ureagenesis in mammals. Despite its relatively low activity, the intestinal
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synthesis of urea from ammonia may be a first line of defence against the potential toxicity of ammonia produced by the extensive intestinal degradation of glutamine and derived from diet and luminal microorganisms. The presence of CPS-I, OCT, ASS and ASL in enterocytes also provides a biochemical basis for synthesizing citrulline and arginine from glutamine by the small intestine [38,39]. The activity of arginase, the final enzyme of the urea cycle, plays a key role in modulating the release by the small intestine of endogenously synthesized and diet-derived arginine into the portal vein [30,37], and therefore, in regulating arginine homeostasis in the body. To the best of our knowledge, there is little or no published information on the compartmentation or kinetics of urea cycle enzymes in the small intestine or enterocytes. A lack of such knowledge has impaired our understanding of arginine metabolism in the gut as well as its role in maintaining arginine homeostasis in mammals. The objective of this study was to determine the compartmentation and kinetics of urea cycle enzymes in enterocytes of the pig, which is widely used as an animal model for studying human intestinal physiology and metabolism [33,38].
2. Materials and methods
2.1. Chemicals L-Amino acids, L-argininosuccinate, D-glucose, ammonium chloride, NaHCO3, bovine serum albumin (BSA, fraction V, essentially fatty-acids free), HEPES, ornithine carbamoyltransferase (from streptococcus faecalis), N-acetylglutamate, ATP, DL-dithiothreitol (DTT), sucrose, EDTA, phenylmethylsulfonyl fluoride, aprotinin, chymostatin, pepstatin, o-phthaldialdehyde (OPA), 3-[N-morpholino]propanesulfonic acid (MOPS), and digitonin were purchased from Sigma (St. Louis, MO). HPLC-grade methanol and water were obtained from Fisher Scientific (Houston, TX).
2.2. Animals and the isolation of jejunum and li6er Pigs were offspring of Yorkshire×Landrace sows and Duroc× Hampshire boars, and were obtained from the Swine Center of Texas A and M University. Piglets were processed (castrated, needle teeth clipped, iron shot and ear notched) by 2 days of age. Piglets were weaned at 21 days of age to a corn and soybeanmeal based diet (20% protein) that met the NRC requirements of all nutrients [35]. At 60 days of age, the jejunum and liver were dissected from anesthetized pigs, as previously described [33]. The experiments were carried out in accordance with the guidelines of the National Research Council for the care and use of
animals, and were approved by Texas A and M University’s Institutional Animal Care and Use Committee.
2.3. Preparation of enterocytes Enterocytes were prepared from the pig jejunum, using oxygenated (95% O2/5% CO2) Ca2 + -free Krebs– Henseleit bicarbonate (KHB) buffer supplemented with 5 mM EDTA, as previously described [33]. Briefly, the jejunum (50 cm) was thoroughly rinsed with 0.9% NaCl to remove its luminal contents. It was then rinsed with 3× 50 ml of oxygenated (95% O2/5% CO2) Ca2 + -free KHB buffer (pH 7.4) containing 20 mM HEPES and 5 mM EDTA. The jejunum was filled with 60 ml of Ca2+free KHB buffer and placed in a flask containing 200 ml of this buffer for incubation at 37oC for 50 min. At the end of the incubation, the jejunum was gently patted with the finger-tips for 1 min, and the lumen was drained into polystyrene tubes. The pellet (enterocytes) was obtained by centrifugation at 400× g for 3 min.
2.4. Preparation of cytosolic and mitochondrial fractions of enterocytes for enzyme assays Enterocytes (40 mg protein) were washed twice with 10 ml of 250 mM sucrose/1 mM EDTA/50 mM potassium phosphate buffer (pH 7.5) containing protease inhibitors (5 mg ml − 1 phenylmethylsulfonyl fluoride, 5 mg ml − 1 aprotinin, 5 mg ml − 1 chymostatin and 5 mg ml − 1 pepstatin), by centrifugation at 400× g and 4oC for 3 min. Cells were homogenized at 4oC in 4 ml of this buffer containing 2.5 mM DTT, and the homogenates were centrifuged at 600×g and 4oC for 15 min. The supernatant was centrifuged at 14000 × g and 4oC for 15 min to obtain the cytosol (supernatant fluid) and mitochondria (pellet). The pellet was washed twice with 2 ml of 250 mM sucrose/1 mM EDTA/50 mM potassium phosphate buffer (pH 7.5) containing 2.5 mM DTT and protease inhibitors (5 mg ml − 1 phenylmethylsulfonyl fluoride, 5 mg ml − 1 aprotinin, 5 mg ml − 1 chymostatin and 5 mg ml − 1 pepstatin), and were then suspended in 1 ml of this buffer. Mitochondria were lysed by three cycles of freezing (in liquid nitrogen) and thawing (at 37oC), and the lysates were centrifuged at 10000×g and 4oC for 15 min. The supernatant was used for enzyme assays.
2.5. Preparation of cytosolic and mitochondrial fractions of enterocytes for measurements of metabolite concentrations Enterocytes were incubated at 37oC for 30 min in the presence of 5 mM glucose, 0.2 mM NH4Cl and plasma concentrations of all amino acids [35]. At the end of the incubation, cells were washed three times with 10 ml of fresh KHB buffer containing 2.5% dialyzed BSA to
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remove extracellular amino acids, which was confirmed by HPLC analysis of amino acids [39]. Cytosolic and mitochondrial fractions were prepared as previously described [40]. Briefly, a 2 ml microcentrifuge tube was filled with 0.2 ml of 1.5 M HClO4 overlaid by a 0.7 ml of inert silicone oil [36]. Enterocyte suspension (0.5 ml) was rapidly mixed with 1.7 ml of an ice-old medium containing 0.25 M sucrose, 20 mM MOPS buffer (pH 7.0), 3 mM EDTA, and 1 mg ml − 1 digitonin. After 40 s, 0.5 ml of the cell mixture was loaded onto the top layer of the microcentrifuge tube for centrifugation (3000×g, 0oC, 20 s). The top layer (cytosolic fraction) and the bottom acid layer (mitochondria) were used for analysis of ammonia, amino acids, carbamoyl phosphate, argininosuccinate and ATP, as previously described [33 – 39]. Substrate concentrations in the cytosol and mitochondria were calculated on the basis of water volume in enterocytes (437958 nl per 106 cells, mean9SEM, n = 10) and mitochondrial matrix volume (1.219 0.28 ml mg − 1 protein, mean9 SEM, n = 10), respectively. Water volume in enterocytes and mitochondrial matrix volume were determined by incubating cells or mitochondria in the presence of 3H2O and [14C]sucrose, as previously described [15,36].
2.6. Measurements of the acti6ities of urea cycle enzymes in the cytosol and mitochondrial fractions of enterocytes The activities of CPS-1, OCT, ASS and ASL were determined at 37oC as previously described [33]. All the enzyme assays were performed at two protein levels and three time points (0, 10 and 20 min). Briefly, the assay mixture for CPS-1 (0.5 ml) consisted of 0.15 M potassium phosphate buffer (pH 7.5), 25 mM ATP, 25 mM MgCl2, 5 mM N-acetylglutamate, 20 mM NH4Cl, 5 mM ornithine, 100 mM NaHCO3, 10 U of added ornithine carbamoyltransferase, and mitochondrial extracts (0.5 mg protein). The reaction mixtures were prepared in 0.15 M potassium phosphate buffer (pH 7.5) and were carefully adjusted to pH 7.5 before starting enzyme reaction. Our CPS-I assay was based on the method developed by Hager and Jones [11] and used by other investigators [18]. In our preliminary studies, we found that the use of phosphate buffer (0.15 M, pH 7.5) decreased CPS-I activity by 18% (n= 6) compared with Tris buffer (0.1 M, pH 7.5). We did not use Tris buffer in CPS-I assay because this buffer contained large amounts of an unknown compound that was eluted closely to the citrulline peak on our HPLC chromatograms, which could result in difficulties in accurate peak integrations. In aqueous solution, free ammonia (NH3) is at equilibrium with ammonium ion (NH4+ ) [8]. At pH 7.5, 37oC, and pKa of 9.2, the ratio of [NH3]/[NH4+ ]
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is 0.02, according the Henderson–Hasselbalch equation: pH=pKa + log ([NH3]/[NH4+ ]) [8]. NH3 and Mg-ATP (rather than NH4+ and ATP) are true substrates for CPS-I. The assay medium for OCT (2.0 ml) contained 0.1 M potassium phosphate buffer (pH 7.5), 15 mM ornithine, 40 mM carbamoyl phosphate, and mitochondrial extracts (0.04 mg protein). Our OCT assay was based on the method of Ceriotti [5]. The ASS assay mixture (0.2 ml) consisted of 75 mM potassium phosphate buffer (pH 7.5), 10 mM citrulline, 5 mM aspartate, 5 mM ATP, 5 mM MgSO4, and cytosolic extracts (0.4 mg protein). Mg-ATP (rather than ATP) is a true substrate for ASS. The ASL assay mixture (40 ml) contained 129 mM sodium phosphate buffer (pH 7.0), 10 mM argininosuccinate, 65 mM EDTA and cytosolic extracts (0.1 mg protein). For arginase assay, a mixture of 100 ml of 10 mM MnCl2 (in 50 mM Tris–HCl buffer, pH 7.5) and 100 ml of mitochondrial or cytosolic extracts (0.5 mg protein) was pre-heated at 55oC for 5 min. After cooling to room temperature, the mixture was incubated for 0, 10 or 20 min with 100 ml of 60 mM arginine (in 50 mM Tris–HCl buffer, pH 7.5). Arginine solution was adjusted to pH 7.5 with 1 N HCl before addition to the assay mixture. Arginase activity was also measured without pre-heating enzyme protein at 55oC before addition of arginine. For all above enzyme assays, the incubation was terminated by addition of 100 ml of 1.5 mM HClO4, and the acidified medium was neutralized with 50 ml of 2 mM K2CO3. The neutralized extracts were used for analysis of citrulline (for CPS-1 and OCT assays), urea and ornithine (for arginase assay), arginine (for ASL assay), and arginine plus argininosuccinate (for ASS assay) as previously described [33]. To assess mitochondrial contamination of the cytosolic fraction, the activity of ornithine aminotransferase (OAT) (a mitochondrial enzyme) in the cytosolic and mitochondrial fractions of enterocytes was determined as previously described [37]. To assess cytosolic contamination of the mitochondrial fraction, the activity of P5C reductase (a cytosolic enzyme in pig enterocytes) in the cytosolic and mitochondrial fractions was also determined as previously described [37]. To assess the capacity for carbamoyl phosphate synthesis in the cytosol by CPS-II (a glutamine-utilizing enzyme) compared with that in mitochondria by CPS-I (an ammonia- utilizing enzyme), CPS-II activity was measured in the cytosol of pig enterocytes as described for CPS-I, except that 20 mM L-glutamine replaced 20 mM NH4Cl in the assay mixture [11]. Protein in the cytosol and mitochondria was determined by a modified Lowry method using BSA as standard [20]. Enzyme activities were expressed on the basis of protein.
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Fig. 1. Activities of carbamoylphosphate synthase-I (CPS-I) at various substrate concentrations in mitochondria of porcine enterocytes. Data are mean9 SEM, n = 5. (A) Varying [NH4+ ] at constant [ATP] (30 mM) and [HCO3− ] (100 mM); increasing [NH4+ ] from 0.5 to 20 mM increased (P B0.05) CPS-I activity. (B) Varying [HCO3− ] at constant [ATP] (30 mM) and [NH4+ ] (20 mM); increasing [HCO3− ] from 5 to 100 mM increased (P B0.05) CPS-I activity. (C) Varying [ATP] at constant [NH4+ ] (20 mM) and [HCO3− ] (100 mM); increasing [ATP] from 1 to 30 mM increased CPS-I activity. CP, carbamoylphosphate.
2.7. Determination of Vmax and Km 6alues for enterocyte urea cycle enzymes The assay of CPS-1 was carried out as described above, except: (1) Varying [HCO3− ] from 5 to 100 mM at constant [ATP] (30 mM) and [NH4+ ] (20 mM); (2) varying NH4+ from 0.5 to 25 mM at constant [ATP] (30 mM) and [HCO3− ] (100 mM); (3) varying [ATP] from 1 to 35 mM at constant [HCO3− ] (100 mM) and [NH + ] (20 mM). The assay of OCT was carried out as described above, except: (1) Varying [ornithine] from 1 to 60 mM at constant [CP] (40 mM); (2) varying [CP] from 1 to 60 mM at constant [ornithine] (15 mM). The assay of ASS was carried out as described above, except: (1) varying [citrulline] from 0.02 to 10 mM at constant [ATP] (5 mM) and [aspartate] (5 mM); (2) varying [aspartate] from 0.025 to 5 mM at constant [ATP] (5 mM) and [citrulline] (10 mM); (3) varying
[ATP] from 0.5 to 25 mM at constant [citrulline] (10 mM) and [aspartate] (5 mM). The assays of ASL and arginase were carried out as described above, except that [argininosuccinate] varied from 0.031 to 10 mM and [arginine] varied from 1 to 30 mM, respectively. Vmax and Km values of enzymes were determined according to the plots of s versus 6 in Figs. 1–5, on the basis of the definition that Vmax is the maximum velocity of reaction and Km is the substrate concentration at which enzyme velocity is half-maximal [9].
2.8. Determination of kinetics of urea cycle enzymes in pig li6er To compare kinetics of urea cycle enzymes between porcine enterocytes and liver, the cytosol and mitochondrial fractions of pig livers were prepared as described above for enterocytes. Vmax and Km values of
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Fig. 2. Activities of ornithine carbamoyltransferase (OCT) at various substrate concentrations in mitochondria of porcine enterocytes. Data are mean9 SEM, n = 5. (A) [Ornithine] varied at constant [CP] (40 mM). Increasing [ornithine] from 1 to 12.5 mM increased OCT activity. (B) [Carbamoylphosphate] varied at constant [ornithine] (15 mM). Increasing [carbamoylphosphate] from 1 to 40 mM increased (P B0.05) OCT activity. Cit, citrulline.
CPS-I and OCT in mitochondria and of ASS, ASL and arginase in the cytosol were determined as described above, except that the amount of cytosolic protein used for arginase assay was 5 mg.
2.9. Statistical analysis Data were analyzed by unpaired t-test for comparison of the cytosol and mitochondrial enzyme activities, or by one-way ANOVA with the Student – Newman– Keuls test for comparison of activities of a given enzyme at various substrate concentrations [27]. Probability values less than 0.05 (P B0.05) were taken to indicate statistical significance.
3. Results
3.1. Acti6ities of urea cycle enzymes in the cytosol and mitochondria of enterocytes
3.2. Acti6ities of OAT, P5C reductase and CPS-II in the cytosol and mitochondria of enterocytes Ornithine aminotransferase activity was found only in the mitochondrial fraction of enterocytes, and was not detected in the cytosolic fraction. The activity of P5C reductase was found only in the cytosolic fraction of enterocytes, and was not detected in the mitochondrial fraction. CPS-II activity in the cytosol of pig enterocytes was 0.2939 0.034 nmol min − 1 per mg protein (mean9 SEM, n= 6), which was much lower than CPS-I activity in pig enterocytes (Table 1).
3.3. Kinetics of urea cycle enzymes in enterocytes Changes in activities of urea cycle enzymes with increasing substrate concentrations are illustrated in Figs. 1–5. Vmax and Km values of urea cycle enzymes are summarized in Table 2.
3.4. Carbamoylphosphate synthase-I These data are summarized in Table 1. In porcine enterocytes, CPS-I and OCT were exclusively located in mitochondria, ASS and ASL were found only in the cytosol, and arginase was present in both the cytosol and mitochondria. When enzyme protein was preheated at 55oC before addition of arginine substrate, arginase activity was 3-fold greater in the cytosol than in mitochondria. However, when arginase assay did not involve pre-heating of enzyme protein at 55oC before addition of arginine, arginase activity was found to be similar between the cytosolic and mitochondrial fractions. Thus, preheating of enzyme protein at 55oC had no effect (P\ 0.05) on cytosolic arginase activity, but markedly decreased (P B0.05) mitochondrial arginase activity.
At constant [ATP] (30 mM) and [HCO3− ] (100 mM), increasing [NH4+ ] from 0.5 to 15 mM increased (PB 0.05) CPS-I activity in a concentration-dependent manner, but there was no further increase in the enzyme activity with increasing [NH4+ ] from 15 to 25 mM (Fig. 1A). At constant [NH4+ ] (20 mM) and [ATP] (30 mM), increasing [HCO3− ] from 5 to 100 mM progressively increased (P B0.05) CPS-I activity, but there was no difference (P\ 0.05) in the enzyme activity between 100 and 140 mM [HCO3− ] (Fig. 1B). At constant [NH4+ ] (20 mM) and [HCO3− ] (100 mM), increasing [ATP] from 1 to 30 mM gradually increased (PB 0.05) CPS-I activity, but there was no further increase (P\ 0.05) in the enzyme activity between 30 and 35 mM [ATP] (Fig. 1C).
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Fig. 3. Activities of argininosuccinate synthase (ASS) at various substrate concentrations in the cytosol of porcine enterocytes. Data are mean9 SEM, n =5. (A) [ATP] varied at constant [citrulline] (10 mM) and [aspartate] (5 mM). Increasing [ATP] from 0.5 to 5 mM increased (PB 0.05) ASS activity. (B) [Aspartate] varied at constant [ATP] (5 mM) and [citrulline] (10 mM). Increasing [aspartate] from 0.025 to 0.25 mM increased (P B0.05) ASS activity. (C) [Citrulline] varied at constant [ATP] (5 mM) and [aspartate] (5 mM). Increasing [citrulline] from 0.02 to 1 mM increased ASS activity. AS, argininosuccinate.
3.5. Ornithine carbamoyltransferase At constant [CP] (40 mM), increasing [ornithine] from 1 to 12.5 mM progressively increased (P B 0.05) OCT activity, but there was no further increase in the enzyme activity with increasing [ornithine] from 12.5 to 15 mM (Fig. 2A). Interestingly, increasing [ornithine] from 15 to 20, 30, 40 and 60 mM at constant [CP] (40 mM) progressively decreased (P B0.05) OCT activity by 45.3, 52.4, 57.7 and 63.5% (n =5), respectively. At constant [ornithine] (15 mM), increasing [CP] from 1 to 40 mM gradually increased (P B0.05) OCT activity, but there was no further increase in the enzyme activity between 40 and 60 mM [CP] (Fig. 2B).
3.6. Argininosuccinate synthase At constant [citrulline] (10 mM) and [aspartate] (5 mM), increasing [ATP] from 0.5 to 5 mM increased (P B 0.05) ASS activity in a concentration-dependent manner, but there was no further increase in the en-
zyme activity between 5 and 6 mM [ATP] (Fig. 3A). Interestingly, increasing [ATP] from 6 to 10, 15 and 25 mM at constant [citrulline] (10 mM) and [aspartate] (5 mM) progressively decreased (P B 0.05) ASS activity by 70.4, 92.5 and 96.0%, respectively. At constant [ATP] (5 mM) and [citrulline] (10 mM), increasing [aspartate] from 0.025 to 0.25 mM increased (PB 0.05) ASS activity in a concentration-dependent manner, but there was no further increase in the enzyme activity between 0.25 and 5 mM aspartate (Fig. 3B). At constant [ATP] (5 mM) and [aspartate] (5 mM), increasing [citrulline] from 0.02 to 1 mM increased (PB 0.05) ASS activity, but there was no difference in the enzyme activity between 1 and 10 mM citrulline (Fig. 3C).
3.7. Argininosuccinate lyase Increasing [argininosuccinate] from 0.031 to 2.5 mM progressively increased (PB0.05) ASL activity in a concentration-dependent manner. There was no difference (P\0.05) in ASL activity between 2.5 and 10 mM argininosuccinate (Fig. 4).
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Table 1 The activities of urea cycle enzymes and ornithine aminotransferasea
Fig. 4. The activities of argininosuccinate lyase (ASL) at various substrate concentrations in the cytosol of porcine enterocytes. Data are mean9 SEM, n = 5. Increasing [argininosuccinate] from 0.031 to 2.5 mM increased (P B0.05) ASL activity. There was no difference (P \0.05) in ASL activity between 2.5 and 10 mM argininosuccinate. Arg, arginine.
3.8. Arginase When arginase assay involved preheating enzyme protein at 55oC before addition of arginine, increasing [arginine] from 1 to 20 mM progressively increased (P B 0.05) cytosolic and mitochondrial arginase activity in a concentration-dependent manner, but there was no difference (P\ 0.05) in the enzyme activity between 20 and 30 mM arginine (Fig. 5A). When enzyme protein was not preheated at 55oC, increasing [arginine] from 1 to 20 mM also progressively increased (P B 0.05) cytosolic and mitochondrial arginase activity in a concentration-dependent manner, and there was no difference (P\ 0.05) in the enzyme activity between the cytosolic and mitochondrial fractions at each arginine concentration used (Fig. 5B). Preheating enzyme protein at 55oC decreased (PB0.05) Vmax value of mitochondrial
Enzyme
Cytosol (nmol min−1 per mg protein)
Mitochondria
CPS-I OCT ASS ASL Arginase (preheated at 55oC) Arginase (no preheating) OAT P5C reductase
ND " ND " 1.91 90.13 3.57 90.24 7.32 9 0.39
6.52 9 0.25 708.2 9 46.7 ND " ND " 2.01 90.17*
7.16 9 0.54
7.28 9 0.65
ND§ 63.7 9 8.2
362.4 9 12.8 ND§
Enzyme activities were measured as described in Section 2. Data are mean 9SEM, n = 6. CPS-1, carbamoylphosphate synthase-1; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; OAT, ornithine aminotransferase; P5C, pyrroline-5-carboxylate; ND, not detected. " Detection limit was 100 pmol ml−1 of sample; § Detection limit was 7 nmol ml−1 of sample. a In the cytosol and mitochondria of porcine enterocytes. * PB0.01: different from the cytosol, as analyzed by unpaired t-test.
arginase but had no effect (P\ 0.05) on the cytosolic enzyme (Table 2). Km values were similar between these two isoforms of arginase (Table 2).
3.9. Concentrations of urea cycle enzyme substrates in pig enterocytes These data are summarized in Table 3. The cytosol of enterocytes contained relatively large amounts of ATP, aspartate and carbamoylphosphate, and to a lesser extent, ammonia, ornithine, citrulline and arginine. There was a small amount of argininosuccinate in the cytosol of enterocytes. Mitochondria contained rela-
Fig. 5. The activities of cytosolic and mitochondrial arginase at various substrate concentrations in porcine enterocytes. Data are mean 9SEM, n=5. (A) Arginase activities were measured by pre-heating enzyme protein at 55°C before addition of arginine substrate. Increasing [arginine] from 1 to 20 mM increased (PB 0.05) arginase activity. (B) Arginase activities were measured without pre-heating enzyme protein before the addition of arginine substrate. Increasing [arginine] from 1 to 20 mM increased (P B0.05) arginase activity.
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Table 2 Vmax and Km of urea cycle enzymes in the cytosol or mitochondria of porcine enterocytes and liver Enzyme
Substrate
Enterocytes Vmax
Km
Vmax
Km
6.409 0.37 6.889 0.43 6.759 0.26
1.34 90.11 58.6 9 2.4 15.2 9 1.4
18.6 90.23* 19.0 9 0.16* 18.3 9 0.21*
1.20 9 0.16 5.77 9 0.49* 1.69 9 0.14*
713.4 9 22.3 699.59 32.1
5.13 90.24 17.1 91.2
915.7 9 68.2* 926.3 9 73.0*
1.58 9 0.12* 0.46 9 0.05*
Mg-ATP Asp Cit
1.849 0.10 1.939 0.07 1.909 0.08
2.86 90.16 0.054 9 0.003 0.15 9 0.01
8.58 90.72* 8.36 90.53* 8.42 90.51*
0.24 9 0.03* 0.031 90.002* 0.068 90.01*
CPS-I (Mit)
NH+ 4 HCO− 3 Mg-ATP
OCT (Mit)
Orn CP
ASS (Cyt)
AS
3.509 0.23
0.63 9 0.04
17.7 90.19*
0.12 9 0.01*
a
Arg
7.13 90.60
7.46 9 0.52
3072 9 256*
3.38 9 0.26*
a
Arg
1.9390.14
7.09 90.58
ASL (Cyt) Arginase (Cyt)
Liver
Arginase (Mit)
—
—
Data are mean 9 SEM in nmol min−1 per mg protein for Vmax and mM for Km; n = 5. CPS-1, carbamoylphosphate synthase-1; OCT, ornithine carbamoyltransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; CP, carbamoyl phosphate; AS, argininosuccinate; Cyt, cytosol; Mit, mitochondria. a Enzyme protein was preheated at 55°C before addition of arginine. * PB0.01: Different from the corresponding value for enterocytes, as analyzed by unpaired t-test.
tively large amounts of ammonia, ATP, aspartate, and to a lesser extent, ornithine, citrulline, arginine and carbamoylphosphate.
3.10. Kinetics of urea cycle enzymes in the li6er Vmax and Km values of CPS-I, OCT, ASS, ASL and arginase are summarized in Table 2. In the liver, arginase had the greatest activity among the urea cycle enzymes, followed by OCT, CPS-I, ASL and ASS. Except for OCT, Vmax values of urea cycles enzymes were much greater (P B 0.01) in the porcine liver than in enterocytes, and vice versa for Km values of these enzymes.
Table 3 Concentrations of urea cycle enzyme substrates in pig enterocytes Substrate
Cytosol
Mitochondria
Ammonia ATP Aspartate Citrulline Arginine Ornithine Argininosuccinate Carbamoylphosphate
0.459 0.06 4.289 0.51 5.569 0.62 0.3690.04 0.8490.09 0.309 0.03 0.0429 0.005 2.26 9 0.19
1.189 0.07* 8.56 9 0.93* 2.419 0.33* 0.529 0.06* 0.759 0.08 0.619 0.07* ND 0.599 0.07*
Data are mean 9 SEM in mM; n= 6. ND, not detectable. Detection limit for amino acid analysis by our HPLC method was 100 pmol ml−1 of sample. * PB0.01: Different from the cytosol value, as analyzed by unpaired t-test.
4. Discussion The results of this study demonstrate that in porcine enterocytes: (1) CPS-I and OCT were exclusively located in mitochondria; (2) ASS and ASL were found only in the cytosol; and (3) arginase was present in both the cytosol and mitochondria (Table 1). To the best of our knowledge, this is the first report of compartmentation of urea cycle enzymes in enterocytes. As in the liver [22], intestinal urea synthesis from ammonia spans two compartments: the cytosol and mitochondria. Among urea cycle enzymes in enterocytes, the activity of OCT was the highest, followed by arginase, CPS-I, ASL and ASS in decreasing order. The cytosolic arginase appears to differ from the mitochondrial enzyme in that the latter, but not the former, is inactivated by pre-heating of enzyme protein at 55oC. Interestingly, when enzyme protein was not preheated at 55oC, arginase activity was equally distributed between the cytosol and mitochondria. Thus, two isoforms of arginase are present in porcine enterocytes, as reported in the rat small intestine on the basis of electrophoretic separation [12]. This is consistent with the recent report that porcine enterocytes contain anionic and non-anionic isoforms of arginase and that preheating enzyme protein (60oC, 15 min) decreased arginase activity in suckling pigs enterocytes [24]. In the study of M’Rabet-Touil et al. [24], it was not known whether cytosolic or mitochondrial arginase in porcine enterocytes was sensitive to heat inactivation because porcine enterocytes were stored at − 80oC and homogenized by sonification, thereby releasing both cytosolic and mitochondrial enzymes into the same supernatant fluid used for the arginase assay.
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To the best of our knowledge, this is the first report of the kinetics of all urea cycle enzymes in enterocytes. Km values of ASS for ATP, aspartate and citrulline were much lower than the concentrations of these substrates in the cytosol of enterocytes, suggesting a relatively high affinity of ASS for its substrates. However, Vmax value of ASS was the lowest among all urea cycle enzymes in enterocytes. Carbamoylphosphate concentrations in mitochondria of enterocytes were much lower than the Km value of CPS-I for this substrate, which may explain a relatively low activity of CPS-I in pig enterocytes. These results suggest that ASS and carbamoylphosphate may be rate-limiting factors in the synthesis of citrulline from glutamine in enterocyte mitochondria. Similarly, Km values of OCT, ASL and arginase for ornithine, argininosuccinate and arginine (Table 2) were much greater than concentrations of these substrates in the cytosol or mitochondria of enterocytes (Table 3), suggesting that rates of fluxes through these enzymes are far from maximal in intact enterocytes. The relatively high Km value of OCT for carbamoylphosphate compared to its intramitochondrial concentrations may explain preferential channelling of extracellular arginine or ornithine into OAT for the synthesis of P5C and therefore of proline in pig enterocytes [37]. Although porcine intestinal OCT activity was inhibited by excess amounts of ornithine (\ 15 mM), as reported for purified porcine liver OCT [16], this inhibition may not be of physiological relevance because a much greater concentration of ornithine is required for enzyme inhibition than ornithine concentration in enterocyte mitochondria (0.81 mM) (Table 3). Substrate inhibition of enzymes was also observed for porcine intestinal ASS when [ATP] was greater than 6 mM. This inhibition is not likely to be physiologically relevant on the basis of ATP concentrations in the cytosol of enterocytes (4.28 mM) (Table 3). Kinetics of hepatic urea cycle enzymes appears to be similar between pigs (Table 2) and rats [22]. However, in pigs, kinetics of urea cycle enzymes differs remarkably between enterocytes and the liver. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values (Table 2). Urea cycle enzymes also differ between porcine enterocytes and liver in their responses to physiological and nutritional alterations. For example, starvation, feeding a high protein diet and glucagon treatment increase both the activities and mRNA levels of all urea cycle enzymes including OCT in the liver [23,28]. In contrast, glucagon treatment has been shown to have no effect on the activities and mRNA levels of intestinal CPS-I or OCT [25]. Also, feeding a high protein diet has been reported to decrease the activity and mRNA level of intestinal OCT [31,32]. The difference in enzyme expressions between the liver and small intestine in response to protein
535
feeding is not restricted to urea cycle enzymes, as feeding a high protein diet increases OAT activity in the liver but decreases the enzyme activity in the small intestine [21]. The enzymological differences between the liver and small intestine in response to physiological and nutritional alterations may be related to the major metabolic functions of these two organs. For example, an increase in dietary protein intake necessitates an increase in the activities of hepatic urea cycle enzymes to remove ammonia produced from amino acid oxidation. On the other hand, in mammals fed a high protein diet, there is no need for increasing intestinal production of citrulline for endogenous synthesis of arginine. The urea cycle enzymes in postweaning pig enterocytes may have dual functions: (1) Synthesizing citrulline from ammonia, HCO3− and ornithine; and (2) converting ammonia, HCO3− and aspartate into urea. Both cytosolic and mitochondrial isoforms of arginase are induced in enterocytes of postweaning pigs (Table 1) [33]. As a result, enterocytes shift from a net synthesis of arginine in neonates [10,38] to a net release of citrulline in postweaning pigs [39]. Because of a low rate of urea synthesis from ammonia in enterocytes compared with the liver [22], we suggest that the presence of enterocyte CPS-I and OCT may function primarily to synthesize citrulline from ammonia, HCO3− and ornithine in postweaning pigs. This suggestion is consistent with the view of Campbell [6] regarding the function of intestinal CPS-I and OCT in ureotelic species. The co-localization of CPS-I and OCT and a high activity of OCT in enterocyte mitochondria favors the synthesis of citrulline from ammonia, HCO3− and ornithine in the pig small intestine, as in the mammalian liver [26]. The location of OAT in mitochondria greatly facilitates the provision of ornithine from glutamine in enterocytes. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes which have a relatively high activity of arginase. Such kinetic properties of intestinal urea cycle enzymes help to maximize the net synthesis of citrulline from glutamine [39], and explain the release of large amounts of citrulline from the small intestine of postweaning pigs [35]. Ornithine aminotransferase and P5C reductase are required for proline synthesis from glutamine and arginine. Glutamine is metabolized to P5C via phosphate-dependent glutaminase and P5C synthase in enterocyte mitochondria [38]. Some of the glutamine-derived P5C is then transported from mitochondria into the cytosol for proline synthesis by P5C reductase. The co-localization of arginase and OAT in enterocyte mitochondria also facilitates the synthesis of P5C from arginine, because the arginine-derived ornithine is efficiently channelled to OAT. Argininederived P5C is then transported from mitochondria
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Fig. 6. Compartmentation of the synthesis of proline from glutamine and arginine in enterocytes. PDG, phosphate-dependent glutaminase; P5C, pyrroline-5-carboxylate; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; NOS, nitric oxide synthase; BH4, tetrahydrobiopterin; NO, nitric oxide.
into the cytosol for proline synthesis. This may help to explain, in part, the much speculated role of mitochondrial arginase in extrahepatic tissues and cells [13]. These compartmentalized reactions for proline synthesis are summarized in Fig. 6. The role of OAT in proline synthesis is substantiated by our finding that an inhibition of this enzyme leads to a decrease in proline synthesis from arginine by 80 – 85% in pig enterocytes [37]. It is worth pointing out that the location of P5C reductase in the cytosol of enterocytes prevents or minimizes a potential recycling of glutamine-derived P5C into proline, thereby ensuring net synthesis of citrulline from glutamine. By regulating the hydrolysis of arginine into ornithine (an inhibitor of P5C synthase [19]), mitochondrial arginase may play an important role in regulating the intestinal synthesis of P5C and therefore of citrulline and proline from glutamine. On the other hand, cytosolic arginase may play a critical role in modulating intracellular concentrations of arginine for other competing arginine-requiring pathways such as NO synthesis (Fig. 6) as in macrophages [29] and endothelial cells [4]. An alteration of NO production may, in turn, contribute to the regulation of intestinal function [1]. Cytosolic arginase may also regulate the provision of
ornithine for ornithine decarboxylase (a cytosolic enzyme) (Fig. 6), the first and key regulatory enzyme in the synthesis of polyamines, thereby regulating the proliferation and differentiation of intestinal epithelial cells [14]. In conclusion, the urea cycle enzymes in porcine enterocytes span two compartments: mitochondria (CPS-I, OCT and arginase) and the cytosol (ASS, ASL and arginase). Except for OCT, Vmax values of urea cycle enzymes are much lower in enterocytes than in the liver, and vice versa for their Km values. The compartmentation and kinetics properties of urea cycle enzymes provide a biochemical basis for explaining net synthesis of large amounts of citrulline from glutamine in enterocytes of postweaning pigs. The presence of arginase in mitochondria and the cytosol may play an important role in regulating the metabolism of proline, nitric oxide and polyamines in enterocytes.
Acknowledgements We thank Wene Yan, Nick E. Flynn, Sean P. Flynn and Darrell A. Knabe for technical assistance, as well as Pat Onstott and Frances Mutscher for secretarial
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support. This research was supported by USDA competitive grants c 94-37206-1100 and c 97-35206-5096 (to G. Wu).
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