422
BIOCHEMICAL SOCIETY TRANSACTIONS
might be expected to accompany the fourfold increase in differential rate of exoprotein formation observed by Abbas-Ali & Coleman (1977). The data presented show that exoprotein formation by Staph. aureus has characteristics consistent with the ‘competition’ model proposed by Coleman et al. (1975) and support the contention that it may be generally applicable to those organisms that secrete exoprotein maximally after the end of exponential growth. Abbas-Ali, B. & Coleman, G. (1977) J. Gen. Microbiol. in the press Brown,S. & Coleman, G. (1975) J. Mol. Biol.96, 345-352 Coleman, G., Brown,S. & Stormonth, D. A. (1975) J. Theor. Biol.52, 143-148
Partial Purification of Two Lithocholic Acid-Binding Proteins from Rat Liver lOOOOOg Supernatants RICHARD C. STRANGE, ROBERT CRAMB and IAlN W. PERCY-ROBB Department of Clinical Chemistry, University of Edinburgh, Royal Infirmary, Edinburgh EH3 9 YW, Scotland, U.K.
Bile acid transport is a quantitatively important aspect of hepatic anion transport that is poorly understood. We have described the presence of binding sites for bile acids (cholic acid, glycocholic acid, chenodeoxycholic acid and lithocholic acid) in rat liver lOOOOOg supernatants (Strange et al., 1976, 1977). The large values both for the dissociation constants of binding (approx. 1 0 - 6 ~and ) the concentration of binding sites (approx. 10-6mol/g of supernatant protein) suggested that these binding components might be involved in transporting bile acids across the hepatocyte. We now describe (a) the purification of lithocholic acid-bindingcomponents and (b) examination of partially purified preparations for glutathione S-transferase activity similar to that of the non-specilic anion-binding protein ligandin (Habig et al., 1974). The purification procedures used are shown in Table 1. Each of the partially purified preparations was examined for lithocholic acid-binding activity by eluting a mixture of the preparation and [14C]lithocholicacid from a Sephadex G-75 column. Radioactive lithocholic acid was eluted with proteins of mol.wt. 40000, and the lithocholic acidbinding activity was expressed as the ratio between the maximum amount of [14C]lithocholic acid (d.p.s./ml) bound to protein and the protein concentration (mg/ml) of that fraction. The partially purified preparations were tested for glutathione S-transferase activity by measuring the conjugation of l-chloro-2,4-dinitrobenzene with glutathione at 340nm (Habig et al., 1974).
Table 1. Purification of the lithocholic acid-binding proteins Volume Protein (mg) (ml) 1600 lOOOOOg supernatant 40 Sephadex G-75 100 500 120 (NH&SO, fractionation 10 50 Calcium phosphate gel 10 10 DEB-Sephadex 12 CM-Sephadex 5.5 Peak (1) 9.0 3.5 peak (2) 10.5
Lithocholic acid 1-Chloro-2,4-dinitrobinding benzene activity (AAlmin (d.p.s./mg) per mg of protein) 1200 27 2000 50 4000 70 6OOo 100 10000 200 13000 8000
250 230 1977
567th MEETING, DURHAM
423
CM-Sephadex chromatography (phosphate buffer, l o r n , pH7.4) resolved the lithocholicacid-bindingmixture obtained fromDEAE-Sephadexinto two fractions, both able to bind lithocholic acid and both having glutathione S-transferase activity. Agarose and polyacrylamide-gel electrophoresis of the fractions obtained from CM-Sephadex chromatography demonstrated the presence of only a single protein-containing band in each fraction. These fractions were further investigated for glutathione S-transferase activity by measuring the conjugation of ethacrynic acid [2,3-dichloro-4-(2-ethylacryloy1)phenoxyacetic acid] with glutathione. Both fractions had this activity, suggesting that one of them is the anion-bindingproteinligandin (Habigetal., 1974). The physiological relevanceof these findings is not clear. The value of the dissociation constant for binding of lithocholic acid to hepatic supernatants suggests a transport function. Ligandin can bind many different anions which are transported into bile by the liver (Levi et al., 1969). Phylogenetic and clinical studies have led to the proposal that ligandin is involved in the hepatic transport of bromosulphophthalein and bilirubin. It seems unlikely, however, that ligandin is involved in the transport of bile acids as well as bilirubin and bromosulphophthalein, since studies have suggested that bile acid transport is mediated by a mechanism different from the other anions. Habig, W. H., Pabst, M. J. Rt Jacoby, W. B. (1974) J. Biol. Chem. 249,7130-7139 Levi, A. J., Gatmaitan, Z. & Arias, I. M. (1969) J. Clin. Invest. 48, 2156-2167 Strange, R. C., Nimmo, I. A. & Percy-Robb, 1. W. (1976) Biochem. J. 156,427433 Strange, R. C., Nimmo, I. A. & Percy-Robb, I. W. (1977) Biochem. J. 162, 659-664
The Effect of Elimination of the Gastrointestinal Flora on the Accumulation of Methylmercuric Chloride by the Rat IAN R. ROWLAND, MARGARET J. DAVIES and PAUL GRASS0 British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, U.K.
When methylmercuric chloride labelled with '03Hg (CH3'03HgCl) was incubated in vitro with the contents of the small intestine or caecum of the rat, the radioactivity was volatilized over a period of 3-4 days (Fig. 1). To determine whether the intestinal metabolismof CH3HgClhas any significancein viuo,we have compared the accumulation of CH3203HgCIby organs of conventional rats and of rats treated with antibiotics to eliminate their intestinal microflora. Of two groups of seven male Wistar rats (135-150g), obtained from a specificpathogen-free breeding colony, one group (control) was given water and autoclaved Spillers Laboratory Small Animal Diet ad lib., and the other group the autoclaved diet and water containing bacitracin, neomycin sulphate and streptomycin sulphate, each at 2mg/ml. Faecal samples were taken each day so that the degree of bacterial colonization of the intestinal tract could be determined. The samples were inoculated on to plates of blood agar and incubated aerobically and anaerobically. The faeces were found to be free of bacteria after 3 days of antibiotic treatment. On days 4-8 and 11-14 inclusive, the rats were given, by stomach tube, 0.5 ml of CHoZo3HgCI in 0.9 % NaCl, such that each rat received nine doses of 13pg (0.03pCi of '03Hg) as CH3HgCI. At 18h after the last treatment, the rats were anaesthetized with diethyl ether and killed by exsanguination. The radioactivity in the kidneys, liver, lung, brain, skeletal muscle, blood and faeces was measured and hence the mercury concentration in the tissues could be calculated (Table 1). The amount of mercury accumulated by the blood, kidneys, brain, muscle and lung was signihntly greater in the animals treated with antibiotics than in the control rats, indicating that the intestinal flora influences the absorption of mercury from
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BIOCHEMICAL SOCIETY TRANSACTIONS
might be expected to accompany the fourfold increase in differential rate of exoprotein formation observed by Abbas-Ali & Coleman (1977). The data presented show that exoprotein formation by Staph. aureus has characteristics consistent with the ‘competition’ model proposed by Coleman et al. (1975) and support the contention that it may be generally applicable to those organisms that secrete exoprotein maximally after the end of exponential growth. Abbas-Ali, B. & Coleman, G. (1977) J. Gen. Microbiol. in the press Brown,S. & Coleman, G. (1975) J. Mol. Biol.96, 345-352 Coleman, G., Brown,S. & Stormonth, D. A. (1975) J. Theor. Biol.52, 143-148
Partial Purification of Two Lithocholic Acid-Binding Proteins from Rat Liver lOOOOOg Supernatants RICHARD C. STRANGE, ROBERT CRAMB and IAlN W. PERCY-ROBB Department of Clinical Chemistry, University of Edinburgh, Royal Infirmary, Edinburgh EH3 9 YW, Scotland, U.K.
Bile acid transport is a quantitatively important aspect of hepatic anion transport that is poorly understood. We have described the presence of binding sites for bile acids (cholic acid, glycocholic acid, chenodeoxycholic acid and lithocholic acid) in rat liver lOOOOOg supernatants (Strange et al., 1976, 1977). The large values both for the dissociation constants of binding (approx. 1 0 - 6 ~and ) the concentration of binding sites (approx. 10-6mol/g of supernatant protein) suggested that these binding components might be involved in transporting bile acids across the hepatocyte. We now describe (a) the purification of lithocholic acid-bindingcomponents and (b) examination of partially purified preparations for glutathione S-transferase activity similar to that of the non-specilic anion-binding protein ligandin (Habig et al., 1974). The purification procedures used are shown in Table 1. Each of the partially purified preparations was examined for lithocholic acid-binding activity by eluting a mixture of the preparation and [14C]lithocholicacid from a Sephadex G-75 column. Radioactive lithocholic acid was eluted with proteins of mol.wt. 40000, and the lithocholic acidbinding activity was expressed as the ratio between the maximum amount of [14C]lithocholic acid (d.p.s./ml) bound to protein and the protein concentration (mg/ml) of that fraction. The partially purified preparations were tested for glutathione S-transferase activity by measuring the conjugation of l-chloro-2,4-dinitrobenzene with glutathione at 340nm (Habig et al., 1974).
Table 1. Purification of the lithocholic acid-binding proteins Volume Protein (mg) (ml) 1600 lOOOOOg supernatant 40 Sephadex G-75 100 500 120 (NH&SO, fractionation 10 50 Calcium phosphate gel 10 10 DEB-Sephadex 12 CM-Sephadex 5.5 Peak (1) 9.0 3.5 peak (2) 10.5
Lithocholic acid 1-Chloro-2,4-dinitrobinding benzene activity (AAlmin (d.p.s./mg) per mg of protein) 1200 27 2000 50 4000 70 6OOo 100 10000 200 13000 8000
250 230 1977
567th MEETING, DURHAM
423
CM-Sephadex chromatography (phosphate buffer, l o r n , pH7.4) resolved the lithocholicacid-bindingmixture obtained fromDEAE-Sephadexinto two fractions, both able to bind lithocholic acid and both having glutathione S-transferase activity. Agarose and polyacrylamide-gel electrophoresis of the fractions obtained from CM-Sephadex chromatography demonstrated the presence of only a single protein-containing band in each fraction. These fractions were further investigated for glutathione S-transferase activity by measuring the conjugation of ethacrynic acid [2,3-dichloro-4-(2-ethylacryloy1)phenoxyacetic acid] with glutathione. Both fractions had this activity, suggesting that one of them is the anion-bindingproteinligandin (Habigetal., 1974). The physiological relevanceof these findings is not clear. The value of the dissociation constant for binding of lithocholic acid to hepatic supernatants suggests a transport function. Ligandin can bind many different anions which are transported into bile by the liver (Levi et al., 1969). Phylogenetic and clinical studies have led to the proposal that ligandin is involved in the hepatic transport of bromosulphophthalein and bilirubin. It seems unlikely, however, that ligandin is involved in the transport of bile acids as well as bilirubin and bromosulphophthalein, since studies have suggested that bile acid transport is mediated by a mechanism different from the other anions. Habig, W. H., Pabst, M. J. Rt Jacoby, W. B. (1974) J. Biol. Chem. 249,7130-7139 Levi, A. J., Gatmaitan, Z. & Arias, I. M. (1969) J. Clin. Invest. 48, 2156-2167 Strange, R. C., Nimmo, I. A. & Percy-Robb, 1. W. (1976) Biochem. J. 156,427433 Strange, R. C., Nimmo, I. A. & Percy-Robb, I. W. (1977) Biochem. J. 162, 659-664
The Effect of Elimination of the Gastrointestinal Flora on the Accumulation of Methylmercuric Chloride by the Rat IAN R. ROWLAND, MARGARET J. DAVIES and PAUL GRASS0 British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS, U.K.
When methylmercuric chloride labelled with '03Hg (CH3'03HgCl) was incubated in vitro with the contents of the small intestine or caecum of the rat, the radioactivity was volatilized over a period of 3-4 days (Fig. 1). To determine whether the intestinal metabolismof CH3HgClhas any significancein viuo,we have compared the accumulation of CH3203HgCIby organs of conventional rats and of rats treated with antibiotics to eliminate their intestinal microflora. Of two groups of seven male Wistar rats (135-150g), obtained from a specificpathogen-free breeding colony, one group (control) was given water and autoclaved Spillers Laboratory Small Animal Diet ad lib., and the other group the autoclaved diet and water containing bacitracin, neomycin sulphate and streptomycin sulphate, each at 2mg/ml. Faecal samples were taken each day so that the degree of bacterial colonization of the intestinal tract could be determined. The samples were inoculated on to plates of blood agar and incubated aerobically and anaerobically. The faeces were found to be free of bacteria after 3 days of antibiotic treatment. On days 4-8 and 11-14 inclusive, the rats were given, by stomach tube, 0.5 ml of CHoZo3HgCI in 0.9 % NaCl, such that each rat received nine doses of 13pg (0.03pCi of '03Hg) as CH3HgCI. At 18h after the last treatment, the rats were anaesthetized with diethyl ether and killed by exsanguination. The radioactivity in the kidneys, liver, lung, brain, skeletal muscle, blood and faeces was measured and hence the mercury concentration in the tissues could be calculated (Table 1). The amount of mercury accumulated by the blood, kidneys, brain, muscle and lung was signihntly greater in the animals treated with antibiotics than in the control rats, indicating that the intestinal flora influences the absorption of mercury from
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