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extracts were chromatographed on plates of silica gel G, with chloroformln-hexane (9: 1, v/v) as developer (Imura et al., 1971), to show that all the extracted radioactivity migrated at the same rate as a CH3HgCI standard. The amount of methylmercury extracted from the tissues by this method varied from 13.9 to 85.9% (Table 1). In model experiments to determine the degree of extraction of CH3203HgC1from tissue homogenates, between 81 and 86% of added CHJZo3HgCIcould be extracted by benzene. Therefore it appears that in brain and skeletal muscle virtually all of the accumulated Hg is in the form of CH3HgCl, whereas in blood, kidney, liver and faeces a substantial part of the Hg is present in other forms, presumably inorganic (Table 1; Norseth & Clarkson, 1971). In the kidney, although not in any other organ tested, the percentage of Hg present as methylmercury was significantly higher in the rats without a gut flora than in the control rats (Table l), which again suggests that the intestinal flora plays some part in the metabolism of CH3HgCl. It must be noted that, although the concentration of Hg in the faeces was much lower in the antibiotic-treated rats than in the controls, the total weight of faeces produced by the former was much greater and hence, in terms of the total amount of Hg excreted, the two groups may not differ significantly.Nevertheless, the percentage of total Hg present as methylmercury in the faeces of the antibiotic-treatedanimals was almost twice that in the faeces of the controls (Table l), thus providing further evidence for the role of the gut flora in metabolizing CH3HgCI. It appears from our results therefore that members of the gastrointestinal flora can metabolize CH3HgCI and convert it into a form that is absorbed from the intestine, or accumulated by tissues, less readily than CH3HgCI itself. Imura, N., Sukegawa, E., Pan, S. K., Hagao, K., Kim, J. Y.,Kwant, T. & Utika, T. (1971) Science 172,1248-1249 Norseth, T. & Clarkson, T. W. (1971) Arch. Enuiron. Health 22, 568-580 Westoo, G. (1966) Acta Chem. Scand. 20,2131-2136
Copper- and Zinc-Binding Protein Fractions in the Soluble Cytoplasm of Rat Tissues DEREK S. NORTON and FRANK W. HEATON Department of Biological Sciences, University of Lancaster, Lancaster LA1 4 YQ,U.K. Copper and zinc occur in the soft tissues of several mammalian species associated with soluble proteins having mol.wts. of about 10000 and 35000 (Evans et al., 1970; Bremner & Marshall, 1974; Bremner, 1976). When the supernatant from rat liver is separated by gel filtration on Sephadex G-75two corresponding fractions are obtained, but copper and zinc are also found associated with protein-containing fractions having mol.wts. of approx. 65000 and over 75000 (Bremner et al., 1973; Alfaro & Heaton, 1974). The present investigationwas therefore undertaken to examine the distribution of copper and zinc among proteins in the supernatants of several rat tissues under conditions that would permit the separation of materials over a wider range of molecular weight. Studies were conducted on weanling (50g) and adult (300-5OOg) Wistar albino rats of both sexes. The liver, small intestine and thigh muscle were homogenized separately in a Potter-Elvehjem homogenizer with a Teflon pestle, in ice-cold 0.075 M-potassium phosphate buffer, pH7.0, to give 50 % (w/v) homogenates. The homogenates were centrifuged at l05000g for 60min and the supernatants stored at -20°C if they could not be fractionated immediately. A portion (4ml) of each supernatant was applied to a column (62cmx2.6cm) of Sephadex G-150, and elution was carried out at 4°C with
VOl. 5
426
BIOCHEMICAL SOCIETY TRANSACTIONS
r
10.8
3 --. 3 0.12 -
- 0.6
0.I6
.-8
U
c)
E
c)
- 0.4
0.08-
8
8
5
9 0 ._ * U
0.04
- 0.2
-
B8 d
JO
0
x Molecular weight
Fig. 1. Distribution of copper (a) and zinc (u) in supernatant fractions of adult male rat liver separated on Sephadex G-150 For further details, see the text.
0.05M-Tris/HC1 buffer, pH7.0, containing 0.1 M-KCI. Fractions of volume 4ml were collected and their Azso values measured automatically to detect protein. The amounts of copper and zinc in the fractions were determined by atomic absorption flame photometry after deproteinizationwith ~ M - H and C ~30% (w/v) trichloroacetic acid. A Dupont 310 Curve Resolver was used to measure the distribution of each metal between the various bands in the elution profile. The column was calibrated by using five proteins of known mol.wt. ranging from 12400 to 240000. The elution profile for liver supernatant from adult male rats (Fig. 1)-showed the presence of five bands containing copper, zinc and protein with mol.wts. of approx. 10000, 22000, 75000, 150000 and 250000 or higher. An intermediate fraction of mol.wt. 42000 containingonly copper and protein was also present. Six similar fractions were found in small intestine and thigh muscle, except that in muscle the 75000-mol.wt. fraction contained only copper and protein, whereas copper, zinc and protein were all present in the 42000-mol.wt. fraction. The qualitative composition of fractions from any one organ was not affected by the age or sex of the rat. Quantitative differences in the distribution of copper and zinc between the fractions were, however, observed in rats of different ages. The differences were particularly marked in the liver, where the proportions of the supernatant copper and zinc associated with the 10000-moI.wt.fraction were much lower in adult animals than in weanling rats. Conversely the proportions of both metals in the 22000-mol.wt. fraction were markedly increased in the older animals and the proportion of zinc in the 75000-mol.wt. fraction also tended to be higher in the liver and small intestine of adult rats. The importance of the different fractions in zinc metabolism was investigated by injecting 20pCi of carrier-free 65ZnC12in 0.9 % NaCl into the tail vein of three adult male rats and killing them at intervals of l0min. 1h and 24 h after the injection. The liver, small intestine and thigh muscle were removed and their supernatants fractionated as described above. The 65Zn in the fractions eluted from the gel-filtration column was measured with a Nuclear Enterprises SR3 y-counter, and related to the total zinc determined by the atomic-absorption technique. The speczc radioactivity of 6sZn was similar in all fractions obtained from small intestine and thigh muscle, but differences were observed in the liver. At lOmin after the injection of 65Zn, the specific radioactivity was highest in the fractions with mol.wts. of about 10000 (Fig. 2), but after 1 h the activity in this area was decreased
1977
567th MEETING, DURHAM
421
3.0 -
2.0
-
1.0
--
280
I
I
I
I
I
I
160
80
40
20
10
0
x Molecular weight
Fig. 2. SpeciJc radioactivity of 65Znin fractions of liver supernatant lOmin (A), 1h ( A ) and 24h (0)after intravenous injection of the isotope The horizontal line (----) represents uniform distribution among stable zinc.
and a secondary peak of activity was noted in the fractions with mol.wts. around 75000. The radioactivity equilibrated most slowly with the 22000-m0l.w. fraction, but after 24h it was almost uniformly distributed among the endogenous zinc. Copper and zinc therefore occur in the soluble cytoplasm of rat tissues associated with a wider range of proteins than was previously recognized. The metals may be bound in a non-selective way to some of these proteins, but the evidence of differences in distribution with age and after the injection of 65Zn indicates that the fractions with mol.wts. of about 10000, 22000 and 75000 are involved in the metabolism of copper and zinc in the liver. The 10000-mol.wt. fraction may contain metallothionein, which has been isolated from the rat (Voelter et al., 1973) in addition to equine and human tissues (Kagi & Vallee, 1961; Pulido et al., 1966), but further work is necessary to investigate the nature of the fractions with mol.wts. of approx. 22000 and 75000. We thank the Science Research Council for the award of a studentship to D. S. N. Alfaro, B. & Heaton, F. W. (1974) Br. J. Nutr. 32, 435-445 Bremner, 1. (1976) Br. J. Nub. 35, 245-252 Bremner, I. & Marshall, R. B. (1974) Br. J. Nurr. 32, 283-291 Bremner, I., Davies, N. T. & Mills, C. F. (1973) Biochem. SOC.Trans. 1, 982-985 Evans, G . W., Majors, P. F. & Cornatzer, W. E. (1970) Biochem. Biophys. Res. Commun. 40, 1142-1 148
Kagi, J. H. R. &Vallee, B. L. (1961)J. Biol. Chem. 236, 2435-2442 Pulido, P., Kagi, J. H. R. & Vallee, B. L. (1966) Biochemistry 5, 1768-1777 Voelter, W., Voetsch, W. & Jung, G. (1973) Eur. J. Biochem. 39, 127-140
VOl. 5
425
extracts were chromatographed on plates of silica gel G, with chloroformln-hexane (9: 1, v/v) as developer (Imura et al., 1971), to show that all the extracted radioactivity migrated at the same rate as a CH3HgCI standard. The amount of methylmercury extracted from the tissues by this method varied from 13.9 to 85.9% (Table 1). In model experiments to determine the degree of extraction of CH3203HgC1from tissue homogenates, between 81 and 86% of added CHJZo3HgCIcould be extracted by benzene. Therefore it appears that in brain and skeletal muscle virtually all of the accumulated Hg is in the form of CH3HgCl, whereas in blood, kidney, liver and faeces a substantial part of the Hg is present in other forms, presumably inorganic (Table 1; Norseth & Clarkson, 1971). In the kidney, although not in any other organ tested, the percentage of Hg present as methylmercury was significantly higher in the rats without a gut flora than in the control rats (Table l), which again suggests that the intestinal flora plays some part in the metabolism of CH3HgCl. It must be noted that, although the concentration of Hg in the faeces was much lower in the antibiotic-treated rats than in the controls, the total weight of faeces produced by the former was much greater and hence, in terms of the total amount of Hg excreted, the two groups may not differ significantly.Nevertheless, the percentage of total Hg present as methylmercury in the faeces of the antibiotic-treatedanimals was almost twice that in the faeces of the controls (Table l), thus providing further evidence for the role of the gut flora in metabolizing CH3HgCI. It appears from our results therefore that members of the gastrointestinal flora can metabolize CH3HgCI and convert it into a form that is absorbed from the intestine, or accumulated by tissues, less readily than CH3HgCI itself. Imura, N., Sukegawa, E., Pan, S. K., Hagao, K., Kim, J. Y.,Kwant, T. & Utika, T. (1971) Science 172,1248-1249 Norseth, T. & Clarkson, T. W. (1971) Arch. Enuiron. Health 22, 568-580 Westoo, G. (1966) Acta Chem. Scand. 20,2131-2136
Copper- and Zinc-Binding Protein Fractions in the Soluble Cytoplasm of Rat Tissues DEREK S. NORTON and FRANK W. HEATON Department of Biological Sciences, University of Lancaster, Lancaster LA1 4 YQ,U.K. Copper and zinc occur in the soft tissues of several mammalian species associated with soluble proteins having mol.wts. of about 10000 and 35000 (Evans et al., 1970; Bremner & Marshall, 1974; Bremner, 1976). When the supernatant from rat liver is separated by gel filtration on Sephadex G-75two corresponding fractions are obtained, but copper and zinc are also found associated with protein-containing fractions having mol.wts. of approx. 65000 and over 75000 (Bremner et al., 1973; Alfaro & Heaton, 1974). The present investigationwas therefore undertaken to examine the distribution of copper and zinc among proteins in the supernatants of several rat tissues under conditions that would permit the separation of materials over a wider range of molecular weight. Studies were conducted on weanling (50g) and adult (300-5OOg) Wistar albino rats of both sexes. The liver, small intestine and thigh muscle were homogenized separately in a Potter-Elvehjem homogenizer with a Teflon pestle, in ice-cold 0.075 M-potassium phosphate buffer, pH7.0, to give 50 % (w/v) homogenates. The homogenates were centrifuged at l05000g for 60min and the supernatants stored at -20°C if they could not be fractionated immediately. A portion (4ml) of each supernatant was applied to a column (62cmx2.6cm) of Sephadex G-150, and elution was carried out at 4°C with
VOl. 5
426
BIOCHEMICAL SOCIETY TRANSACTIONS
r
10.8
3 --. 3 0.12 -
- 0.6
0.I6
.-8
U
c)
E
c)
- 0.4
0.08-
8
8
5
9 0 ._ * U
0.04
- 0.2
-
B8 d
JO
0
x Molecular weight
Fig. 1. Distribution of copper (a) and zinc (u) in supernatant fractions of adult male rat liver separated on Sephadex G-150 For further details, see the text.
0.05M-Tris/HC1 buffer, pH7.0, containing 0.1 M-KCI. Fractions of volume 4ml were collected and their Azso values measured automatically to detect protein. The amounts of copper and zinc in the fractions were determined by atomic absorption flame photometry after deproteinizationwith ~ M - H and C ~30% (w/v) trichloroacetic acid. A Dupont 310 Curve Resolver was used to measure the distribution of each metal between the various bands in the elution profile. The column was calibrated by using five proteins of known mol.wt. ranging from 12400 to 240000. The elution profile for liver supernatant from adult male rats (Fig. 1)-showed the presence of five bands containing copper, zinc and protein with mol.wts. of approx. 10000, 22000, 75000, 150000 and 250000 or higher. An intermediate fraction of mol.wt. 42000 containingonly copper and protein was also present. Six similar fractions were found in small intestine and thigh muscle, except that in muscle the 75000-mol.wt. fraction contained only copper and protein, whereas copper, zinc and protein were all present in the 42000-mol.wt. fraction. The qualitative composition of fractions from any one organ was not affected by the age or sex of the rat. Quantitative differences in the distribution of copper and zinc between the fractions were, however, observed in rats of different ages. The differences were particularly marked in the liver, where the proportions of the supernatant copper and zinc associated with the 10000-moI.wt.fraction were much lower in adult animals than in weanling rats. Conversely the proportions of both metals in the 22000-mol.wt. fraction were markedly increased in the older animals and the proportion of zinc in the 75000-mol.wt. fraction also tended to be higher in the liver and small intestine of adult rats. The importance of the different fractions in zinc metabolism was investigated by injecting 20pCi of carrier-free 65ZnC12in 0.9 % NaCl into the tail vein of three adult male rats and killing them at intervals of l0min. 1h and 24 h after the injection. The liver, small intestine and thigh muscle were removed and their supernatants fractionated as described above. The 65Zn in the fractions eluted from the gel-filtration column was measured with a Nuclear Enterprises SR3 y-counter, and related to the total zinc determined by the atomic-absorption technique. The speczc radioactivity of 6sZn was similar in all fractions obtained from small intestine and thigh muscle, but differences were observed in the liver. At lOmin after the injection of 65Zn, the specific radioactivity was highest in the fractions with mol.wts. of about 10000 (Fig. 2), but after 1 h the activity in this area was decreased
1977
567th MEETING, DURHAM
421
3.0 -
2.0
-
1.0
--
280
I
I
I
I
I
I
160
80
40
20
10
0
x Molecular weight
Fig. 2. SpeciJc radioactivity of 65Znin fractions of liver supernatant lOmin (A), 1h ( A ) and 24h (0)after intravenous injection of the isotope The horizontal line (----) represents uniform distribution among stable zinc.
and a secondary peak of activity was noted in the fractions with mol.wts. around 75000. The radioactivity equilibrated most slowly with the 22000-m0l.w. fraction, but after 24h it was almost uniformly distributed among the endogenous zinc. Copper and zinc therefore occur in the soluble cytoplasm of rat tissues associated with a wider range of proteins than was previously recognized. The metals may be bound in a non-selective way to some of these proteins, but the evidence of differences in distribution with age and after the injection of 65Zn indicates that the fractions with mol.wts. of about 10000, 22000 and 75000 are involved in the metabolism of copper and zinc in the liver. The 10000-mol.wt. fraction may contain metallothionein, which has been isolated from the rat (Voelter et al., 1973) in addition to equine and human tissues (Kagi & Vallee, 1961; Pulido et al., 1966), but further work is necessary to investigate the nature of the fractions with mol.wts. of approx. 22000 and 75000. We thank the Science Research Council for the award of a studentship to D. S. N. Alfaro, B. & Heaton, F. W. (1974) Br. J. Nutr. 32, 435-445 Bremner, 1. (1976) Br. J. Nub. 35, 245-252 Bremner, I. & Marshall, R. B. (1974) Br. J. Nurr. 32, 283-291 Bremner, I., Davies, N. T. & Mills, C. F. (1973) Biochem. SOC.Trans. 1, 982-985 Evans, G . W., Majors, P. F. & Cornatzer, W. E. (1970) Biochem. Biophys. Res. Commun. 40, 1142-1 148
Kagi, J. H. R. &Vallee, B. L. (1961)J. Biol. Chem. 236, 2435-2442 Pulido, P., Kagi, J. H. R. & Vallee, B. L. (1966) Biochemistry 5, 1768-1777 Voelter, W., Voetsch, W. & Jung, G. (1973) Eur. J. Biochem. 39, 127-140
VOl. 5
