Research in Veterinary Science 1992, 53, 47-51
Tissue distribution of monomeric glutathione peroxidase in broiler chicks S. MIYAZAKI, Y. MOTOI, Feed Safety Research Division, National Institute of Animal Health, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan
It is known that there are two kinds of enzymes which show glutathione peroxidase activity, 'classical' glutathione peroxidase and glutathione Stransferase. Recently, a third enzyme was found, monomeric glutathione peroxidase, in broiler chick fiver cytosolic fraction. To gain an insight into the possible physiological role of the monomeric glutathione peroxidase, the distribution of this enzyme in other broiler tissues was studied. The monomeric glutathione peroxidase was found in all tissues examined and in erythrocytes. The percentage of the total glutathione peroxidase activity accounted for by the monomeric enzyme ranged from 4 per cent in erythrocytes to 28 per cent in liver. Livers from three avian and two mammalian species contained the monomeric glutathione peroxidase. The contribution of the monomeric enzyme to total glutathione peroxidase activity with cumene hydroperoxide was high in poultry livers, while only trace monomeric glutathione peroxidase activity was found in mammalian livers. Chick monomeric glutathione peroxidase showed high activity toward phospholipid hydroperoxide. Thus, monomeric glutathione peroxidase might be an important enzyme in reducing membrane lipid hydroperoxides in birds. G L U T A T H I O N E peroxidase catalyses the reduction of peroxides using reduced glutathione as a hydrogen source. Until recently, this activity was thought to be associated with two enzymes, namely 'classical' glutathione peroxidase (EC 1..11.1.9) and glutathione S-transferase (Ec 2.5.1.18). As selenocysteine residues are essential components of classical glutathione peroxidase (Se-6sH-Px), its activity is eliminated from liver by feeding a selenium deficient diet (Hafeman et al 1974). However, selenium independent glutathione peroxidase (non-Se-GsH-Px), glutathione
S-transferase, is not diminished by selenium deficiency (Lawrence and Burk 1976). Non-Se-muPx does not use hydrogen peroxide well as a substrate but readily reacts with organic hydroperoxides. Recently, a third glutathione peroxidase was found which reduces both hydrogen peroxide and cumene hydroperoxide in broiler chick liver (Miyazaki 1991). This enzyme (M-GSH-PX) is a monomeric protein of 18-5 kDa (SDS-PAGE) and is active toward phospholipid hydroperoxide in the presence of a detergent (authors' unpublished data). The high activity toward phospholipid hydroperoxide suggests the importance of M6SH-Px in the protection of membrane phospholipid from oxidative stress. In this study, the tissue distribution of M-GSHPx is examined, together with that of Se-GSH-Px and non-Se-GsH-Px, in broiler chicks. The occurrence of this activity in the livers of quails, ducks, cattle and rats was also studied. Materials and methods
Chemicals Highly purified NADPH(yeast) and glutathione reductase (yeast) were obtained from Oriental Yeast, Japan and reduced glutathione and 2mercaptoethanol from Wako Pure Chemicals, Japan. All other chemicals were commercial analytical grades.
Preparation of samples Four-week-old male broiler chicks that had been reared on an additive-free diet (sD; Nippon Formula Feed Manufacturing, Japan), were killed by bleeding and their livers were perfused 47
48
S. MiyazakL Y. Motoi
with ice-cold saline. Subsequently their livers, kidneys, duodena, testes and brains were collected for study. They were frozen in liquid nitrogen and kept at -70°C until the time of analysis. Thawed organs were homogenised in three volumes of 0.25 M sucrose containing 5 mM 2-mercaptoethanol using a Teflon homogeniser. In the case of the duodenum, cells were scraped from the mucosal surface with a razor blade and then homogenised like the other organs. Homogenates were centrifuged at 100,000 g for 60 minutes and the cytosolic fractions were collected. Liver samples of other species were prepared in the same way as chick tissues. Heparinised blood was collected by cardiac puncture and plasma was separated by centrifugation. Erythrocytes were obtained by washing the residual blood cells twice with saline. Equal volumes of distilled water were added to obtain a haemolysate that was centrifuged at 100,000 g for 60 minutes. The supernatant was used as a soluble fraction of the erythrocyte. The plasma and erythrocyte soluble fraction were mixed with 2-mercaptoethanol (final 5 mM) and kept at 4°C for 30 minutes before gel permeation chromatographic analyses. Cytosolic fractions and plasma from two or three individuals were pooled and submitted to the following analyses. Determination of enzyme activity Glutathione peroxidase activity, using either hydrogen peroxide or cumene hydroperoxide as substrates, was assayed by the coupled assay system of Lawrence and Burk (1976). In erythrocytes, all haemoglobin was converted to cyanmethaemoglobin by Drabkin's reagent (Paglia and Valentine 1967). Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene was determined according to Habig et al (1974). All enzyme reactions were performed at 25°C. Protein concentration was estimated with the Bio-Rad protein assay kit using bovine gamma globulin as a standard. Gel permeation chromatography Cytosolic preparations were passed through a 0.45 grn filter, and then injected onto a Superose 12 HR 10/30 column (Pharrnacia) and eluted with 50 mM potassium phosphate, 150 mM potassium chloride, 0.5 mM EDTA, 5 mM 2-mercap-
toethanol, pH 7.6 at a flow rate of 0.5 ml rain -1. The eluates were collected in 30 second fractions. Identification of the three enzymes was based on the elution volumes of the enzymes, the presence of glutathione peroxidase activity towards hydrogen peroxide and the presence of glutathione Stransferase activity. The proportion of the activities of the three enzymes to the total glutathione peroxidase activity were determined using cumene hydroperoxide as a substrate. Results
Tissue distribution As reported previously (Miyazaki 1991), chick liver cytosolic glutathione peroxidase activity was separated into three peaks by gel permeation chromatography (Fig la). Only the second peak showed glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene (data not shown). The relative molecular weights and enzyme activities indicated that the first peak was Se-GSH-Px and the second non-Se-GSH-Px. The third peak was the M-~SH-Px enzyme previously described by the authors. The proportions of the three enzyme activities to total liver glutathione peroxidase were approximately 30 per cent, 42 per cent and 28 per cent, respectively (Table 1). The activities of the three enzymes were different in different tissues and the results of typical TABLE 1 : Three kinds of glutathione peroxidase activities in the soluble traction of chick tissues
Tissue
Total*
Liver
103
Kidney
118
Duodenum
106
Brain
23.8
Testis
69.3
Plasma
15.3
Erythrocytes
16.8
Activity (nmol min-1 mg-1) Se non-Se 31,0 (30-1%) 36.6 (30.9%) 10.4 (9.81%) 8.97 (37.7%) 39.3 (56.7%) 15-3
M
43.4 28-7 ( 4 2 - 1 % ) (27.9%) 62-7 19.0 ( 5 3 - 0 % ) (16.1%) 74.9 20.7 ( 7 0 . 7 % ) (19,5%) 10"8 4"00 ( 4 5 " 4 % ) (16"8%) 20.4 9-63 ( 2 9 . 4 % ) (13.9%)
(1o0%) 13.3 (78.9%)
2.82 (16,8%)
0.727 (4.33%)
*Total glutathione peroxidase activity is the cytosolic glutathione peroxidase activity toward cumene hydroperoxide Se Selenium dependent glutathione peroxidase non-Se Selenium independent glutathione peroxidase M Monomeric glutathione peroxidase
49
Monomeric glutathione peroxidase in chicks
chromatograms are shown in Table 1 and Fig 1. In the chick samples examined, the total glutathione peroxidase activity ranged from 118 nmol min-lmg -t in kidney to 15.3 nmol min 1 rag-1 in plasma. In all tissues except plasma (Ffg 1d), glutathione peroxidase activity was separated into three peaks although the chromatographic profiles were different from each other. With plasma only one broad peak was active with both hydrogen peroxide and cumene hydroperoxide.
Although the apparent molecular weight of this peak was almost the same as non-Se-asH-Px, this enzyme was considered a kind of Se-GSH-Px because of its activity toward hydrogen peroxide. In terms of percentage of total glutathione peroxidase activity, Se-asu-Px activity was high in plasma and erythrocytes, intermediate in testis, brain, kidney and liver, and low in duodenum (Fig 1). In erythrocytes, about 80 per cent of total activity was Se-GSI4-Px. As for non-Se-GsN-
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Tissue distribution of monomeric glutathione peroxidase in broiler chicks S. MIYAZAKI, Y. MOTOI, Feed Safety Research Division, National Institute of Animal Health, 3-1-1, Kannondai, Tsukuba, Ibaraki 305, Japan
It is known that there are two kinds of enzymes which show glutathione peroxidase activity, 'classical' glutathione peroxidase and glutathione Stransferase. Recently, a third enzyme was found, monomeric glutathione peroxidase, in broiler chick fiver cytosolic fraction. To gain an insight into the possible physiological role of the monomeric glutathione peroxidase, the distribution of this enzyme in other broiler tissues was studied. The monomeric glutathione peroxidase was found in all tissues examined and in erythrocytes. The percentage of the total glutathione peroxidase activity accounted for by the monomeric enzyme ranged from 4 per cent in erythrocytes to 28 per cent in liver. Livers from three avian and two mammalian species contained the monomeric glutathione peroxidase. The contribution of the monomeric enzyme to total glutathione peroxidase activity with cumene hydroperoxide was high in poultry livers, while only trace monomeric glutathione peroxidase activity was found in mammalian livers. Chick monomeric glutathione peroxidase showed high activity toward phospholipid hydroperoxide. Thus, monomeric glutathione peroxidase might be an important enzyme in reducing membrane lipid hydroperoxides in birds. G L U T A T H I O N E peroxidase catalyses the reduction of peroxides using reduced glutathione as a hydrogen source. Until recently, this activity was thought to be associated with two enzymes, namely 'classical' glutathione peroxidase (EC 1..11.1.9) and glutathione S-transferase (Ec 2.5.1.18). As selenocysteine residues are essential components of classical glutathione peroxidase (Se-6sH-Px), its activity is eliminated from liver by feeding a selenium deficient diet (Hafeman et al 1974). However, selenium independent glutathione peroxidase (non-Se-GsH-Px), glutathione
S-transferase, is not diminished by selenium deficiency (Lawrence and Burk 1976). Non-Se-muPx does not use hydrogen peroxide well as a substrate but readily reacts with organic hydroperoxides. Recently, a third glutathione peroxidase was found which reduces both hydrogen peroxide and cumene hydroperoxide in broiler chick liver (Miyazaki 1991). This enzyme (M-GSH-PX) is a monomeric protein of 18-5 kDa (SDS-PAGE) and is active toward phospholipid hydroperoxide in the presence of a detergent (authors' unpublished data). The high activity toward phospholipid hydroperoxide suggests the importance of M6SH-Px in the protection of membrane phospholipid from oxidative stress. In this study, the tissue distribution of M-GSHPx is examined, together with that of Se-GSH-Px and non-Se-GsH-Px, in broiler chicks. The occurrence of this activity in the livers of quails, ducks, cattle and rats was also studied. Materials and methods
Chemicals Highly purified NADPH(yeast) and glutathione reductase (yeast) were obtained from Oriental Yeast, Japan and reduced glutathione and 2mercaptoethanol from Wako Pure Chemicals, Japan. All other chemicals were commercial analytical grades.
Preparation of samples Four-week-old male broiler chicks that had been reared on an additive-free diet (sD; Nippon Formula Feed Manufacturing, Japan), were killed by bleeding and their livers were perfused 47
48
S. MiyazakL Y. Motoi
with ice-cold saline. Subsequently their livers, kidneys, duodena, testes and brains were collected for study. They were frozen in liquid nitrogen and kept at -70°C until the time of analysis. Thawed organs were homogenised in three volumes of 0.25 M sucrose containing 5 mM 2-mercaptoethanol using a Teflon homogeniser. In the case of the duodenum, cells were scraped from the mucosal surface with a razor blade and then homogenised like the other organs. Homogenates were centrifuged at 100,000 g for 60 minutes and the cytosolic fractions were collected. Liver samples of other species were prepared in the same way as chick tissues. Heparinised blood was collected by cardiac puncture and plasma was separated by centrifugation. Erythrocytes were obtained by washing the residual blood cells twice with saline. Equal volumes of distilled water were added to obtain a haemolysate that was centrifuged at 100,000 g for 60 minutes. The supernatant was used as a soluble fraction of the erythrocyte. The plasma and erythrocyte soluble fraction were mixed with 2-mercaptoethanol (final 5 mM) and kept at 4°C for 30 minutes before gel permeation chromatographic analyses. Cytosolic fractions and plasma from two or three individuals were pooled and submitted to the following analyses. Determination of enzyme activity Glutathione peroxidase activity, using either hydrogen peroxide or cumene hydroperoxide as substrates, was assayed by the coupled assay system of Lawrence and Burk (1976). In erythrocytes, all haemoglobin was converted to cyanmethaemoglobin by Drabkin's reagent (Paglia and Valentine 1967). Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene was determined according to Habig et al (1974). All enzyme reactions were performed at 25°C. Protein concentration was estimated with the Bio-Rad protein assay kit using bovine gamma globulin as a standard. Gel permeation chromatography Cytosolic preparations were passed through a 0.45 grn filter, and then injected onto a Superose 12 HR 10/30 column (Pharrnacia) and eluted with 50 mM potassium phosphate, 150 mM potassium chloride, 0.5 mM EDTA, 5 mM 2-mercap-
toethanol, pH 7.6 at a flow rate of 0.5 ml rain -1. The eluates were collected in 30 second fractions. Identification of the three enzymes was based on the elution volumes of the enzymes, the presence of glutathione peroxidase activity towards hydrogen peroxide and the presence of glutathione Stransferase activity. The proportion of the activities of the three enzymes to the total glutathione peroxidase activity were determined using cumene hydroperoxide as a substrate. Results
Tissue distribution As reported previously (Miyazaki 1991), chick liver cytosolic glutathione peroxidase activity was separated into three peaks by gel permeation chromatography (Fig la). Only the second peak showed glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene (data not shown). The relative molecular weights and enzyme activities indicated that the first peak was Se-GSH-Px and the second non-Se-GSH-Px. The third peak was the M-~SH-Px enzyme previously described by the authors. The proportions of the three enzyme activities to total liver glutathione peroxidase were approximately 30 per cent, 42 per cent and 28 per cent, respectively (Table 1). The activities of the three enzymes were different in different tissues and the results of typical TABLE 1 : Three kinds of glutathione peroxidase activities in the soluble traction of chick tissues
Tissue
Total*
Liver
103
Kidney
118
Duodenum
106
Brain
23.8
Testis
69.3
Plasma
15.3
Erythrocytes
16.8
Activity (nmol min-1 mg-1) Se non-Se 31,0 (30-1%) 36.6 (30.9%) 10.4 (9.81%) 8.97 (37.7%) 39.3 (56.7%) 15-3
M
43.4 28-7 ( 4 2 - 1 % ) (27.9%) 62-7 19.0 ( 5 3 - 0 % ) (16.1%) 74.9 20.7 ( 7 0 . 7 % ) (19,5%) 10"8 4"00 ( 4 5 " 4 % ) (16"8%) 20.4 9-63 ( 2 9 . 4 % ) (13.9%)
(1o0%) 13.3 (78.9%)
2.82 (16,8%)
0.727 (4.33%)
*Total glutathione peroxidase activity is the cytosolic glutathione peroxidase activity toward cumene hydroperoxide Se Selenium dependent glutathione peroxidase non-Se Selenium independent glutathione peroxidase M Monomeric glutathione peroxidase
49
Monomeric glutathione peroxidase in chicks
chromatograms are shown in Table 1 and Fig 1. In the chick samples examined, the total glutathione peroxidase activity ranged from 118 nmol min-lmg -t in kidney to 15.3 nmol min 1 rag-1 in plasma. In all tissues except plasma (Ffg 1d), glutathione peroxidase activity was separated into three peaks although the chromatographic profiles were different from each other. With plasma only one broad peak was active with both hydrogen peroxide and cumene hydroperoxide.
Although the apparent molecular weight of this peak was almost the same as non-Se-asH-Px, this enzyme was considered a kind of Se-GSH-Px because of its activity toward hydrogen peroxide. In terms of percentage of total glutathione peroxidase activity, Se-asu-Px activity was high in plasma and erythrocytes, intermediate in testis, brain, kidney and liver, and low in duodenum (Fig 1). In erythrocytes, about 80 per cent of total activity was Se-GSI4-Px. As for non-Se-GsN-
(a) P-150
l
E
E
"5
60
E 40
100
Z" ~
