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Effect of propargylglycine on synthesis of glutathione in mice
Nutrition Research 21 (2001) 1373–1381 www.elsevier.com/locate/nutres
Effect of propargylglycine on synthesis of glutathione in mice Sang K. Kim, Ph.D., Young C. Kim, Ph.D.* College of Pharmacy, Seoul National University, San 56 –1 Shinirim-Dong, Kwanak-Ku, Seoul 151–742, Korea Received 20 March 2001; received in revised form 14 August 2001; accepted 16 August 2001
Abstract Effects of propargylglycine (PPG) treatment on the hepatic glutathione (GSH) synthesis were examined in adult male ICR mice. Administration of PPG (200 mole/kg, ip) to mice resulted in a complete inhibition of the hepatic cystathionine ␥-lyase (C␥L) activity measured in cytosol fraction for 40 hr after the treatment. A single injection of PPG rapidly reduced the hepatic GSH levels, which appeared to be sustained at least for 40 hr. The GSH concentration in plasma was significantly decreased for 20 hr, but recovered to the control level in 40 hr. Renal GSH levels did not appear to be changed by PPG treatment. The cysteine concentrations in liver, kidney and plasma were also decreased by PPG. The effect of PPG pretreatment was examined in mice challenged with methionine (1 mmole/kg, po), the sulfur donor in the transsulfuration pathway. Methionine administration elevated S-adenosylmethionine (SAM) and GSH concentrations in liver significantly when measured 3 hr following the treatment. In PPG pretreated mice the hepatic SAM level was increased, however, elevation of GSH by methionine was inhibited completely suggesting that the supply of cysteine from the methionine cycle for GSH synthesis in liver was blocked by PPG. The results show that generation of cysteine in the transsulfuration pathway has a critical role in the hepatic synthesis of GSH, and PPG can be used as an effective tool for study of sulfur-containing amino acid metabolism in liver. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Propargylglycine; Methionine; Glutathione; Cysteine; Transsulfuration
* Corresponding author. Tel.: ⫹1-82-02-880-7852; fax: ⫹1-82-02-872-1795. E-mail address: [email protected] (Y. C. Kim) 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 3 4 4 - X
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1. Introduction Glutathione (GSH; ␥-glutamylcysteinylglycine) is a tripeptide found in all mammalian tissues. GSH serves diverse vital functions including detoxifying electrophiles, scavenging free radicals, maintaining the essential thiol status of proteins, providing a storage and transfer vehicle for cysteine [1,2]. GSH is synthesized intracellularly by the consecutive actions of ␥-glutamylcysteine synthetase (GCS) and GSH synthetase. GCS is regulated by feedback inhibition of GSH and availability of its precursor, cysteine [3,4,5]. The transsulfuration of methionine, other than diet, is the source of cysteine, which transfers the sulfur of methionine to serine to form cysteine [6,7]. The first step in the methionine metabolism is the formation of S-adenosylmethionine (SAM) that is catalyzed by methionine adenosyltransferase. SAM serves as the methyl donor for biological methylation reactions and the co-product of transmethylation, S-adenosylhomocysteine (SAH), is hydrolyzed to yield homocysteine and adenosine. The transsulfuration of homocysteine to cysteine via cystathionine is mediated by the activities of cystathionine -synthase (CS) and cystathionine ␥-lyase (C␥L). The liver has one of the highest organ contents of GSH and is unique in two aspects of GSH synthesis. First, it converts methionine to cysteine, the essential building block for GSH, through the transsulfuration pathway [8,9]. Second, it exports GSH into plasma and bile at a rate that accounts for nearly all of its biosynthesis [10]. Plasma GSH, derived almost entirely from sinusoidal efflux of hepatic GSH, is taken up by kidney and other organs for a source of cysteine, but not by the liver. Therefore, the liver plays a central role in a complex interorgan homeostasis of sulfur-containing amino acid metabolism [6,11]. Propargylglycine (PPG) was first reported by Abeles and Walsh [12] to inactivate the liver C␥L in mice. The mechanism proposed for the inactivation involved enzymatic conversion of the PPG at the active site to a reactive allene [13]. However, with regard to its effect on GSH levels controversial observations have been obtained. In mice a single intraperitoneal administration of PPG (5 mole/mouse) led to a decrease in hepatic GSH to 40% of the normal level which was maintained for one day [14]. On the other hand hepatic GSH levels were not altered although the C␥L activity was inhibited by greater than 90% in rats injected with PPG for three consecutive days (50 mole/ kg/day) [15]. In the present study the dynamics of GSH concentrations in liver, kidney and plasma was determined in mice treated with PPG. It was aimed to examine the significance of the transsulfuration pathway in cysteine supply for the hepatic GSH synthesis by employing the C␥L inhibitor.
2. Methods and meterials 2.1. Animals and treatments Adult male ICR mice (Animal Breeding Center, Seoul National University) were used throughout the study. The use of animals was in compliance with the guidelines established by the Animal Care Committee of this institute. Animals were housed in temperature (22 ⫾
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2°C) and humidity (55 ⫾ 5%) controlled rooms with a 12-hr light/dark cycle (Light: 0700 –1900, dark 1900 – 0700) for at least one week prior to use. Laboratory chow and tap water were allowed ad libitum. PPG and methionine were dissolved in 0.9% saline. Animals were fasted in stainless-steel wire-bottomed cages for 12 hr before PPG treatment (200 mole/kg, ip). Mice were treated with methionine (1 mmole/kg, po) 4 hr after PPG administration. The fasting was maintained until sacrifice. 2.2. Chemicals Drugs and chemicals such as NADPH, GSSG reductase, methionine, propargylglycine, S-adenosylmethionine iodide salt, GSH, cysteine and homoserine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol was obtained from Merck (Darmstadt, Germany). All other chemicals and solvents were reagent grade or better. 2.3. Assays Mice were killed by decapitation and the liver and kidney were homogenized in a fourfold volume of cold 1 M perchloric acid. The denatured protein was removed by centrifugation at 10,000 g for 10 min. Blood samples were collected into a centrifuge tube containing 5 mole NaEDTA; after immediate centrifugation, the plasma was quickly mixed with 0.5 volume 10% sulfosalicylic acid. Total GSH concentration was determined using an enzymatic recycling method of Griffith [16] or a HPLC separation/fluorometric detection method of Neuschwander-Tetri and Roll [17]. HPLC was performed using a PU-980 pump (Jasco Co., Tokyo, Japan) equipped with a fluorescence detector (FP-920, Jasco Co.) and a C18 reversed phase column (Waters Co., Milford, MA, USA). Cysteine levels were estimated by the acid-ninhydrin method [18]. For determination of SAM the method of She et al. [19] was employed. The supernatant sample was directly applied to a HPLC system consisted of a PU-980 pump, a UV/VIS detector (UV-975, Jasco Co.) and a TSK-GEL ODS-80TM (4.6 ⫻ 250 mm) column (Tosoh Co., Tokyo, Japan). For measurement of the C␥L activity the liver was homogenized in a three-fold volume of an ice-cold buffer consisting of 0.154 M KCl/50 mM Tris-HCl and 1 mM EDTA (pH 7.4). The homogenate was centrifuged at 10,000 g for 20 min and the supernatant fraction was further centrifuged at 104,000 g for 60 min. The 104,000 g supernatant fraction was used to determine the enzyme activities. Protein was determined by the method of Lowry et al. [20]. The activity of C␥L was determined by a modification of the method of Matsuo and Greenberg [21]. The reaction mixture of 1.0 ml contained 32 mM homoserine, 0.05 mM pyridoxal 5-phosphate, 7.5 mM 2-mercaptoethanol, 7.0 mM EDTA, 0.1 M potassium phosphate buffer (pH 7.5) and 1 mg protein of enzyme solution. The incubation was carried at 37 °C for 30 min. The amount of ␣-ketobutyrate, which was formed during the enzymatic reaction, was quantified with 2,4-dinitrophenylhydrazone in alkaline solution spectrophotometrically. Color development was measured at 510 nm.
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Fig. 1. Effect of PPG (200 mole/kg, ip) on hepatic C␥L activity. Value given is the mean ⫾ SE for more than 6 mice. Number in parentheses is percent of the enzyme activity in the control mice.
3. Results and discussion PPG is one of enzyme inhibitors known as suicide inactivators. These substrate analogs inactivate the enzymes irreversibly by covalent interaction with the active site as a result of enzymatic formation of a chemically reactive intermediate [13]. Beatty and Reed [8] showed that the presence of 1 mM PPG in the medium inhibited 35S incorporation into hepatocyte GSH from [35S]methionine. A single injection of PPG (5 mole/mouse) or three consecutive daily intraperitoneal injections of PPG (50 mole/kg) to rats also led to a complete inactivation of the hepatic C␥L [14,15]. Although PPG has been shown to be an irreversible inhibitor of C␥L both in vivo and in vitro, the effect of PPG on hepatic GSH levels is controversial. Treatment of mice with a single dose of PPG resulted in reduction of hepatic GSH to 40% of the control levels [14]. But hepatic GSH was not altered by three consecutive daily injections of PPG to rats [15]. These results suggest the possibility that in vivo inhibition of hepatic C␥L induced by PPG is not complete and/or quantitative contribution to cysteine supply for GSH synthesis by the transsulfuration pathway is minor. In this study the activity of hepatic C␥L was measured for 40 hr after a single PPG injection (200 mole/kg, ip) to mice (Fig. 1). PPG treatment caused an almost complete inhibition of the C␥L activity in the cytosolic fraction. The activity was shown to be stable in the control fasting mice for the period examined in this study. The PPG treatment induced significant alterations in the tissue and plasma levels of GSH (Fig. 2). The hepatic GSH was reduced to approximately 70% of the level measured at the time of PPG injection (t ⫽ 0 hr) for 40 hr. However, the GSH level of the control mice was markedly lowered at t ⫽ 40 hr, probably due to the prolonged fast forced to the animals starting from 12 hr prior to the treatment. There was not a significant difference in the hepatic
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Fig. 2. Effect of PPG (200 mole/kg, ip) on GSH in liver (A), kidney (B) and plasma (C). Value given is the mean ⫾ SE for more than 6 mice. *,**,***Significantly different from the control (P⬍0.05, 0.01, 0.001, respectively, Student’s t-test).
GSH level between the two groups at this time point. The plasma GSH concentration was decreased by PPG at t ⫽ 8 hr and 20 hr in accordance with the changes in the liver. In contrast to liver or plasma, the renal GSH level was not altered by PPG during the whole period. The Effect of PPG on the concentrations of cysteine, the essential building block for GSH, is shown in Fig. 3. In PPG treated mice the hepatic cysteine level was decreased progres-
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Fig. 3. Effect of PPG (200 mole/kg, ip) on cysteine concentrations in liver (A), kidney (B) and plasma (C). Value given is the mean ⫾ SE for more than 6 mice. *,**Significantly different from the control (P⬍0.05, 0.01, Student’s t-test).
sively. The renal cysteine concentration was also lower at t ⫽ 20 hr. The plasma cysteine concentration was rapidly decreased at ⫽ 4 hr, but recovered to the control level at t ⫽ 8 hr. The plasma concentration of cystine, the predominant form in plasma, was also reduced from 93.2 ⫾ 3.6 to 80.1 ⫾ 4.2 nmole/ml plasma 4 hr after PPG treatment. The cysteine concentrations in tissues and plasma were gradually reduced in the control animals, which seemed to be associated with prolonged fasting.
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Table 1 Effect of methionine and PPG on hepatic SAM and GSH levels Group
Control Methionine PPG PPG ⫹ Methionine
SAM
GSH
nmole/g liver
mole/g liver
76.3 ⫾ 1.2 99.0 ⫾ 8.5* 73.4 ⫾ 2.3 90.8 ⫾ 3.0**
4.59 ⫾ 0.09 7.16 ⫾ 0.24*** 2.85 ⫾ 0.26 2.97 ⫾ 0.09
Mice treated with methionine (1 mmole/kg, po) 4 hr after PPG (200 mole/kg, ip). Three hr following methionine treatment animals were sacrificed for the assay. Each value represents the mean ⫾SE for 4 mice. *, **, *** Significantly different from each corresponding control (P ⬍ 0.05, 0.01, 0.001, respectively, Student’s t-test).
The effect of PPG was examined in mice injected with a dose of methionine (1 mmole/kg, po), the sulfur donor for the transsulfuration pathway (Table 1). The concentrations of SAM (an intermediate product in the methionine cycle) and GSH (the final product in the transsulfuration pathway) were siginificantly elevated by methionine treatment. PPG pretreatment did not affect the increase in SAM induced by methionine, however, the increase in GSH was prevented suggesting that the enzymatic reaction(s) mediating transsulfuration from methionine to cysteine was blocked. In the present study a single injection of PPG was shown to induce an almost complete blockade of the generation of cysteine in the transsulfuration pathway. This conclusion is supported by both in vivo and in vitro observations. The activity of C␥L, the enzyme mediating generation of cysteine from cystathionine in the transsulfuration pathway, was inhibited for 40 hr when determined in the hepatic cytosolic fraction of mice pretreated with the enzyme inactivator. Also the hepatic levels of cysteine and GSH were all suppressed in mice by PPG treatment. In liver the availability of cysteine is regulated by a balance between the rate of supply and the rate of metabolism to GSH, taurine and inorganic sulfate. Hepatic cysteine is derived from methionine via the transsulfuration pathway or supplied directly through cysteine uptake mediated by amino acid transport systems [7,22]. The degree of contribution of these two suppliers is unclear. However, the results in this study indicate that cysteine generation in the transsulfuration pathway has an important role in maintaining the hepatic cysteine availability and that, when the supply from the metabolic reaction is inhibited, a significant reduction in hepatic GSH contents results. This view is further supported by results from the experiment treating the animals with a bolus dose of methionine. This sulfur amino acid increased the SAM and GSH concentrations in liver indicating that production of both the intermediate and the final product of the transsulfuration pathway was enhanced by providing the sulfur donor for the metabolic reaction. PPG administration did not affect the methionine cycle as evidenced by the unchanged SAM levels either in the control or in the methionine treated mice, but the hepatic GSH levels were reduced significantly by PPG. Methionine injected into the mice pretreated with PPG failed to affect the concentration of GSH in liver suggesting that supply of the sulfur amino acid precursor from the methionine cycle had been blocked completely by this enzyme inactivator. Decrease in the plasma GSH levels was observed from t ⫽ 8 hr to 20 hr following PPG
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administration. The sinusoidal GSH transport is a carrier-mediated saturable process, the apparent Michaelis-Menten constant of which is reported to be approximately 3.2 mole/g liver [6]. In the present study hepatic GSH was reduced to a level similar to the MichaelisMenten constant for GSH efflux. Also the decrease in plasma GSH was preceded by the reduction of this endogenous tripeptide in liver, suggesting that the GSH decrease in plasma was accounted by the decrease in liver. A single injection of PPG (200 mole/kg, ip) did not alter the renal GSH levels whereas the cysteine levels appeared to be decreased. It is known that kidney has the highest organ contents of cysteine, well above the Michaelis-Menten constant of GCS [23,24], the key enzyme for GSH synthesis. Furthermore, the renal GCS activity per tissue weight was measured to be six to seven fold higher than that of liver [25]. The discrepancy between the effect of PPG on renal GSH and that of cysteine could be explained by these observations. In summary the present study showed that a single administration of PPG to mice led to an almost complete blockade of cysteine synthesis in the transsulfuration pathway, without altering the methionine cycle. It is indicated that cysteine synthesis in the metabolic pathway has a critical role in cysteine supply for GSH synthesis in liver as evidenced by the marked reduction of GSH and cysteine level in liver and plasma. PPG was shown to be an effective tool for limiting the cysteine supply via the methionine cycle, and therefore, the use of this enzyme inactivator can be found in the study of sulfur-containing amino acid metabolism in liver. Acknowledgments This work was supported by a grant from the Ministry of Health and Welfare (HMP-00CD-02-0004). References [1] Meister A, Anderson M E. Glutathione. Annu Rev Biochem 1983;52:711– 60. [2] DeLeve L, Kaplowitz N. Importance and regulation of hepatic GSH. Semin Liver Dis 1990;10:251– 66. [3] Richman PG, Meister A. Regulation of ␥-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 1975;250:1422– 6. [4] Huang CS, Chang LS, Anderson ME, Meister A. Catalytic and regulatory properties of the heavy subunit of rat kidney ␥-glutamylcysteine synthetase. J Biol Chem 1993;268:19,675– 80. [5] Tateishi N, Higashi T, Shinya S, Naruse A, Sakamoto Y. Studies on the regulation of glutathione level in rat liver. J Biochem 1974;75:93–103. [6] Kaplowitz N, Aw TY, Ookhtens M. The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol 1985;25:715– 44. [7] Lu SC. Regulation of hepatic glutathione synthesis. Semin Liver Dis 1998;18:331– 43. [8] Beatty PW, Reed DJ. Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes. Arch Biochem Biophys 1980;204:80 –7. [9] Reed DJ, Orrenius S. The role of methionine in glutathione biosynthesis by isolated hepatocytes. Biochem Biophys Res Commun 1977;77:1257– 64. [10] Lauterburg BH, Adams JD, Mitchell JR. Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover. Hepatology 1984;4:586 –90.
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[11] Ooktens M, Kaplowitz N. Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine. Semin Liver Dis 1998;18:313–29. [12] Abeles RH, Walsh C. Acetylenic enzyme inactivators. Inactivation of ␥-cystathionase, in Vitro and in vivo by propargylglycine. J Am Chem Soc 1973;95:6124 –5. [13] Washtien W, Abeles RH. Mechanism of inactivation of ␥-cystathionase by the acetylenic substrate analogue propargylglycine. Biochemistry 1977;16:2485–91. [14] Shinozuka S, Tanase S, Morino Y. Metabolic consequences of affinity labeling of cystathionase and alanine aminotransferase by L-parpargylglycine in vivo. Eur J Biochem 1982;124:377– 82. [15] Houghton CB, Cherian MG. Effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat. J Biochem Toxicol 1991;6:221– 8. [16] Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl pyridine. Anal Biochem 1980;106:207–12. [17] Neuschwander-Tetri BA, Roll FJ. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct. Anal Biochem 1989; 179:236 – 41. [18] Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 1967;104:627–33. [19] She QB, Nagao I, Hayakawa T, Tsuge H. A simple HPLC method for the determination of S-adenosylmethionine, and S-adenosylhomocysteine in rat tissues: the effect of vitamin B6 deficiency on these concentrations in rat liver. Biochem Biophys Res Commun 1994;205:1748 –54. [20] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951;193:265–75. [21] Matsuo Y, Greenberg DM. A crystalline enzyme that cleaves homoserine, and cystathionine. I. isolation procedure, and some physicochemical properties. J Biol Chem 1957;230:545– 60. [22] Deneke SM, Fanburg B. Regulation of cellular glutathione. Am J Physiol 1989;257:L163–73. [23] Lu SC, Ge JL, Kuhlenkamp J, Kaplowitz N. Insulin and glucocorticoid dependence of hepatic gammaglutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo. J Clin Invest 1992;90:524 –532. [24] Sun WM, Huang ZZ, Lu SC. Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation. Biochem J 1996;320:321–328. [25] Kim S, Wilson JJ, Allen KG, Clarke SD. Suppression of renal gamma-glutamylcysteine synthetase expression in dietary copper deficiency. Biochim Biophys Acta 1996;1313:89 –94.
Effect of propargylglycine on synthesis of glutathione in mice Sang K. Kim, Ph.D., Young C. Kim, Ph.D.* College of Pharmacy, Seoul National University, San 56 –1 Shinirim-Dong, Kwanak-Ku, Seoul 151–742, Korea Received 20 March 2001; received in revised form 14 August 2001; accepted 16 August 2001
Abstract Effects of propargylglycine (PPG) treatment on the hepatic glutathione (GSH) synthesis were examined in adult male ICR mice. Administration of PPG (200 mole/kg, ip) to mice resulted in a complete inhibition of the hepatic cystathionine ␥-lyase (C␥L) activity measured in cytosol fraction for 40 hr after the treatment. A single injection of PPG rapidly reduced the hepatic GSH levels, which appeared to be sustained at least for 40 hr. The GSH concentration in plasma was significantly decreased for 20 hr, but recovered to the control level in 40 hr. Renal GSH levels did not appear to be changed by PPG treatment. The cysteine concentrations in liver, kidney and plasma were also decreased by PPG. The effect of PPG pretreatment was examined in mice challenged with methionine (1 mmole/kg, po), the sulfur donor in the transsulfuration pathway. Methionine administration elevated S-adenosylmethionine (SAM) and GSH concentrations in liver significantly when measured 3 hr following the treatment. In PPG pretreated mice the hepatic SAM level was increased, however, elevation of GSH by methionine was inhibited completely suggesting that the supply of cysteine from the methionine cycle for GSH synthesis in liver was blocked by PPG. The results show that generation of cysteine in the transsulfuration pathway has a critical role in the hepatic synthesis of GSH, and PPG can be used as an effective tool for study of sulfur-containing amino acid metabolism in liver. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Propargylglycine; Methionine; Glutathione; Cysteine; Transsulfuration
* Corresponding author. Tel.: ⫹1-82-02-880-7852; fax: ⫹1-82-02-872-1795. E-mail address: [email protected] (Y. C. Kim) 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 3 4 4 - X
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1. Introduction Glutathione (GSH; ␥-glutamylcysteinylglycine) is a tripeptide found in all mammalian tissues. GSH serves diverse vital functions including detoxifying electrophiles, scavenging free radicals, maintaining the essential thiol status of proteins, providing a storage and transfer vehicle for cysteine [1,2]. GSH is synthesized intracellularly by the consecutive actions of ␥-glutamylcysteine synthetase (GCS) and GSH synthetase. GCS is regulated by feedback inhibition of GSH and availability of its precursor, cysteine [3,4,5]. The transsulfuration of methionine, other than diet, is the source of cysteine, which transfers the sulfur of methionine to serine to form cysteine [6,7]. The first step in the methionine metabolism is the formation of S-adenosylmethionine (SAM) that is catalyzed by methionine adenosyltransferase. SAM serves as the methyl donor for biological methylation reactions and the co-product of transmethylation, S-adenosylhomocysteine (SAH), is hydrolyzed to yield homocysteine and adenosine. The transsulfuration of homocysteine to cysteine via cystathionine is mediated by the activities of cystathionine -synthase (CS) and cystathionine ␥-lyase (C␥L). The liver has one of the highest organ contents of GSH and is unique in two aspects of GSH synthesis. First, it converts methionine to cysteine, the essential building block for GSH, through the transsulfuration pathway [8,9]. Second, it exports GSH into plasma and bile at a rate that accounts for nearly all of its biosynthesis [10]. Plasma GSH, derived almost entirely from sinusoidal efflux of hepatic GSH, is taken up by kidney and other organs for a source of cysteine, but not by the liver. Therefore, the liver plays a central role in a complex interorgan homeostasis of sulfur-containing amino acid metabolism [6,11]. Propargylglycine (PPG) was first reported by Abeles and Walsh [12] to inactivate the liver C␥L in mice. The mechanism proposed for the inactivation involved enzymatic conversion of the PPG at the active site to a reactive allene [13]. However, with regard to its effect on GSH levels controversial observations have been obtained. In mice a single intraperitoneal administration of PPG (5 mole/mouse) led to a decrease in hepatic GSH to 40% of the normal level which was maintained for one day [14]. On the other hand hepatic GSH levels were not altered although the C␥L activity was inhibited by greater than 90% in rats injected with PPG for three consecutive days (50 mole/ kg/day) [15]. In the present study the dynamics of GSH concentrations in liver, kidney and plasma was determined in mice treated with PPG. It was aimed to examine the significance of the transsulfuration pathway in cysteine supply for the hepatic GSH synthesis by employing the C␥L inhibitor.
2. Methods and meterials 2.1. Animals and treatments Adult male ICR mice (Animal Breeding Center, Seoul National University) were used throughout the study. The use of animals was in compliance with the guidelines established by the Animal Care Committee of this institute. Animals were housed in temperature (22 ⫾
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2°C) and humidity (55 ⫾ 5%) controlled rooms with a 12-hr light/dark cycle (Light: 0700 –1900, dark 1900 – 0700) for at least one week prior to use. Laboratory chow and tap water were allowed ad libitum. PPG and methionine were dissolved in 0.9% saline. Animals were fasted in stainless-steel wire-bottomed cages for 12 hr before PPG treatment (200 mole/kg, ip). Mice were treated with methionine (1 mmole/kg, po) 4 hr after PPG administration. The fasting was maintained until sacrifice. 2.2. Chemicals Drugs and chemicals such as NADPH, GSSG reductase, methionine, propargylglycine, S-adenosylmethionine iodide salt, GSH, cysteine and homoserine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol was obtained from Merck (Darmstadt, Germany). All other chemicals and solvents were reagent grade or better. 2.3. Assays Mice were killed by decapitation and the liver and kidney were homogenized in a fourfold volume of cold 1 M perchloric acid. The denatured protein was removed by centrifugation at 10,000 g for 10 min. Blood samples were collected into a centrifuge tube containing 5 mole NaEDTA; after immediate centrifugation, the plasma was quickly mixed with 0.5 volume 10% sulfosalicylic acid. Total GSH concentration was determined using an enzymatic recycling method of Griffith [16] or a HPLC separation/fluorometric detection method of Neuschwander-Tetri and Roll [17]. HPLC was performed using a PU-980 pump (Jasco Co., Tokyo, Japan) equipped with a fluorescence detector (FP-920, Jasco Co.) and a C18 reversed phase column (Waters Co., Milford, MA, USA). Cysteine levels were estimated by the acid-ninhydrin method [18]. For determination of SAM the method of She et al. [19] was employed. The supernatant sample was directly applied to a HPLC system consisted of a PU-980 pump, a UV/VIS detector (UV-975, Jasco Co.) and a TSK-GEL ODS-80TM (4.6 ⫻ 250 mm) column (Tosoh Co., Tokyo, Japan). For measurement of the C␥L activity the liver was homogenized in a three-fold volume of an ice-cold buffer consisting of 0.154 M KCl/50 mM Tris-HCl and 1 mM EDTA (pH 7.4). The homogenate was centrifuged at 10,000 g for 20 min and the supernatant fraction was further centrifuged at 104,000 g for 60 min. The 104,000 g supernatant fraction was used to determine the enzyme activities. Protein was determined by the method of Lowry et al. [20]. The activity of C␥L was determined by a modification of the method of Matsuo and Greenberg [21]. The reaction mixture of 1.0 ml contained 32 mM homoserine, 0.05 mM pyridoxal 5-phosphate, 7.5 mM 2-mercaptoethanol, 7.0 mM EDTA, 0.1 M potassium phosphate buffer (pH 7.5) and 1 mg protein of enzyme solution. The incubation was carried at 37 °C for 30 min. The amount of ␣-ketobutyrate, which was formed during the enzymatic reaction, was quantified with 2,4-dinitrophenylhydrazone in alkaline solution spectrophotometrically. Color development was measured at 510 nm.
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Fig. 1. Effect of PPG (200 mole/kg, ip) on hepatic C␥L activity. Value given is the mean ⫾ SE for more than 6 mice. Number in parentheses is percent of the enzyme activity in the control mice.
3. Results and discussion PPG is one of enzyme inhibitors known as suicide inactivators. These substrate analogs inactivate the enzymes irreversibly by covalent interaction with the active site as a result of enzymatic formation of a chemically reactive intermediate [13]. Beatty and Reed [8] showed that the presence of 1 mM PPG in the medium inhibited 35S incorporation into hepatocyte GSH from [35S]methionine. A single injection of PPG (5 mole/mouse) or three consecutive daily intraperitoneal injections of PPG (50 mole/kg) to rats also led to a complete inactivation of the hepatic C␥L [14,15]. Although PPG has been shown to be an irreversible inhibitor of C␥L both in vivo and in vitro, the effect of PPG on hepatic GSH levels is controversial. Treatment of mice with a single dose of PPG resulted in reduction of hepatic GSH to 40% of the control levels [14]. But hepatic GSH was not altered by three consecutive daily injections of PPG to rats [15]. These results suggest the possibility that in vivo inhibition of hepatic C␥L induced by PPG is not complete and/or quantitative contribution to cysteine supply for GSH synthesis by the transsulfuration pathway is minor. In this study the activity of hepatic C␥L was measured for 40 hr after a single PPG injection (200 mole/kg, ip) to mice (Fig. 1). PPG treatment caused an almost complete inhibition of the C␥L activity in the cytosolic fraction. The activity was shown to be stable in the control fasting mice for the period examined in this study. The PPG treatment induced significant alterations in the tissue and plasma levels of GSH (Fig. 2). The hepatic GSH was reduced to approximately 70% of the level measured at the time of PPG injection (t ⫽ 0 hr) for 40 hr. However, the GSH level of the control mice was markedly lowered at t ⫽ 40 hr, probably due to the prolonged fast forced to the animals starting from 12 hr prior to the treatment. There was not a significant difference in the hepatic
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Fig. 2. Effect of PPG (200 mole/kg, ip) on GSH in liver (A), kidney (B) and plasma (C). Value given is the mean ⫾ SE for more than 6 mice. *,**,***Significantly different from the control (P⬍0.05, 0.01, 0.001, respectively, Student’s t-test).
GSH level between the two groups at this time point. The plasma GSH concentration was decreased by PPG at t ⫽ 8 hr and 20 hr in accordance with the changes in the liver. In contrast to liver or plasma, the renal GSH level was not altered by PPG during the whole period. The Effect of PPG on the concentrations of cysteine, the essential building block for GSH, is shown in Fig. 3. In PPG treated mice the hepatic cysteine level was decreased progres-
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Fig. 3. Effect of PPG (200 mole/kg, ip) on cysteine concentrations in liver (A), kidney (B) and plasma (C). Value given is the mean ⫾ SE for more than 6 mice. *,**Significantly different from the control (P⬍0.05, 0.01, Student’s t-test).
sively. The renal cysteine concentration was also lower at t ⫽ 20 hr. The plasma cysteine concentration was rapidly decreased at ⫽ 4 hr, but recovered to the control level at t ⫽ 8 hr. The plasma concentration of cystine, the predominant form in plasma, was also reduced from 93.2 ⫾ 3.6 to 80.1 ⫾ 4.2 nmole/ml plasma 4 hr after PPG treatment. The cysteine concentrations in tissues and plasma were gradually reduced in the control animals, which seemed to be associated with prolonged fasting.
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Table 1 Effect of methionine and PPG on hepatic SAM and GSH levels Group
Control Methionine PPG PPG ⫹ Methionine
SAM
GSH
nmole/g liver
mole/g liver
76.3 ⫾ 1.2 99.0 ⫾ 8.5* 73.4 ⫾ 2.3 90.8 ⫾ 3.0**
4.59 ⫾ 0.09 7.16 ⫾ 0.24*** 2.85 ⫾ 0.26 2.97 ⫾ 0.09
Mice treated with methionine (1 mmole/kg, po) 4 hr after PPG (200 mole/kg, ip). Three hr following methionine treatment animals were sacrificed for the assay. Each value represents the mean ⫾SE for 4 mice. *, **, *** Significantly different from each corresponding control (P ⬍ 0.05, 0.01, 0.001, respectively, Student’s t-test).
The effect of PPG was examined in mice injected with a dose of methionine (1 mmole/kg, po), the sulfur donor for the transsulfuration pathway (Table 1). The concentrations of SAM (an intermediate product in the methionine cycle) and GSH (the final product in the transsulfuration pathway) were siginificantly elevated by methionine treatment. PPG pretreatment did not affect the increase in SAM induced by methionine, however, the increase in GSH was prevented suggesting that the enzymatic reaction(s) mediating transsulfuration from methionine to cysteine was blocked. In the present study a single injection of PPG was shown to induce an almost complete blockade of the generation of cysteine in the transsulfuration pathway. This conclusion is supported by both in vivo and in vitro observations. The activity of C␥L, the enzyme mediating generation of cysteine from cystathionine in the transsulfuration pathway, was inhibited for 40 hr when determined in the hepatic cytosolic fraction of mice pretreated with the enzyme inactivator. Also the hepatic levels of cysteine and GSH were all suppressed in mice by PPG treatment. In liver the availability of cysteine is regulated by a balance between the rate of supply and the rate of metabolism to GSH, taurine and inorganic sulfate. Hepatic cysteine is derived from methionine via the transsulfuration pathway or supplied directly through cysteine uptake mediated by amino acid transport systems [7,22]. The degree of contribution of these two suppliers is unclear. However, the results in this study indicate that cysteine generation in the transsulfuration pathway has an important role in maintaining the hepatic cysteine availability and that, when the supply from the metabolic reaction is inhibited, a significant reduction in hepatic GSH contents results. This view is further supported by results from the experiment treating the animals with a bolus dose of methionine. This sulfur amino acid increased the SAM and GSH concentrations in liver indicating that production of both the intermediate and the final product of the transsulfuration pathway was enhanced by providing the sulfur donor for the metabolic reaction. PPG administration did not affect the methionine cycle as evidenced by the unchanged SAM levels either in the control or in the methionine treated mice, but the hepatic GSH levels were reduced significantly by PPG. Methionine injected into the mice pretreated with PPG failed to affect the concentration of GSH in liver suggesting that supply of the sulfur amino acid precursor from the methionine cycle had been blocked completely by this enzyme inactivator. Decrease in the plasma GSH levels was observed from t ⫽ 8 hr to 20 hr following PPG
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administration. The sinusoidal GSH transport is a carrier-mediated saturable process, the apparent Michaelis-Menten constant of which is reported to be approximately 3.2 mole/g liver [6]. In the present study hepatic GSH was reduced to a level similar to the MichaelisMenten constant for GSH efflux. Also the decrease in plasma GSH was preceded by the reduction of this endogenous tripeptide in liver, suggesting that the GSH decrease in plasma was accounted by the decrease in liver. A single injection of PPG (200 mole/kg, ip) did not alter the renal GSH levels whereas the cysteine levels appeared to be decreased. It is known that kidney has the highest organ contents of cysteine, well above the Michaelis-Menten constant of GCS [23,24], the key enzyme for GSH synthesis. Furthermore, the renal GCS activity per tissue weight was measured to be six to seven fold higher than that of liver [25]. The discrepancy between the effect of PPG on renal GSH and that of cysteine could be explained by these observations. In summary the present study showed that a single administration of PPG to mice led to an almost complete blockade of cysteine synthesis in the transsulfuration pathway, without altering the methionine cycle. It is indicated that cysteine synthesis in the metabolic pathway has a critical role in cysteine supply for GSH synthesis in liver as evidenced by the marked reduction of GSH and cysteine level in liver and plasma. PPG was shown to be an effective tool for limiting the cysteine supply via the methionine cycle, and therefore, the use of this enzyme inactivator can be found in the study of sulfur-containing amino acid metabolism in liver. Acknowledgments This work was supported by a grant from the Ministry of Health and Welfare (HMP-00CD-02-0004). References [1] Meister A, Anderson M E. Glutathione. Annu Rev Biochem 1983;52:711– 60. [2] DeLeve L, Kaplowitz N. Importance and regulation of hepatic GSH. Semin Liver Dis 1990;10:251– 66. [3] Richman PG, Meister A. Regulation of ␥-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 1975;250:1422– 6. [4] Huang CS, Chang LS, Anderson ME, Meister A. Catalytic and regulatory properties of the heavy subunit of rat kidney ␥-glutamylcysteine synthetase. J Biol Chem 1993;268:19,675– 80. [5] Tateishi N, Higashi T, Shinya S, Naruse A, Sakamoto Y. Studies on the regulation of glutathione level in rat liver. J Biochem 1974;75:93–103. [6] Kaplowitz N, Aw TY, Ookhtens M. The regulation of hepatic glutathione. Annu Rev Pharmacol Toxicol 1985;25:715– 44. [7] Lu SC. Regulation of hepatic glutathione synthesis. Semin Liver Dis 1998;18:331– 43. [8] Beatty PW, Reed DJ. Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes. Arch Biochem Biophys 1980;204:80 –7. [9] Reed DJ, Orrenius S. The role of methionine in glutathione biosynthesis by isolated hepatocytes. Biochem Biophys Res Commun 1977;77:1257– 64. [10] Lauterburg BH, Adams JD, Mitchell JR. Hepatic glutathione homeostasis in the rat: efflux accounts for glutathione turnover. Hepatology 1984;4:586 –90.
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