557th MEETING, LIVERPOOL
683
i.e. oestrone sulphate, dehydroepiandrosterone sulphate, bromosulphophthalein and bilirubin, bound by aminoazodye-binding protein A, are also bound by ligandin, another carcinogen-binding protein widely distributed in the rat (Ketterer et al., 1967; Litwack et al., 1971). It is possible that these two binding proteins are functionally related. We thank the Cancer Research Campaign for a generous grant. Goodman, D. S. (1958) J. Am. Chem. SOC.80,3892-3898 Ketterer, B., Ross-Mansell, P. & Whitehead, J. K. (1967) Biochem. J. 103,316-324 Ketterer, B., Beale, D., Litwack, G. & Hackney, J. F. (1971) Chem.-Biol. Interact. 3,285-286 Litwack, G., Ketterer, B. & Arias, I. M. (1971) Nufure (London) 234,466-467 Mishkin, S . , Stein, L., Gatmaitan, 2. & Arias, I. M. (1972) Biochem. Biophys. Res. Commun. 47,997-1003
Mishkin, S. & Turcotte, R. (1974a) Biochem. Biophys. Res. Commun. 57,918-926 Mishkin, S . & Turcotte, R. (19746) Biochem. Biophys. Res. Commun. 60,376-381 Ockner, R. K., Manning, J. A., Poppenhausen,R. B. & Ho, W. K. L. (1972) Science 177.56-58 Rosenthal, H. E., Pietrzak, E., Slaunwhite, W. R. & Sandberg, A. A. (1972)J. Clin. Endocrinol. 34,805-81 3
Rudman, D., Bixler, T.J. & Del Rio, A. E. (1971) J. Phurmacol. Exp. Ther. 176,261-272
The Effect of Increasing Phenylalanine Concentration on PhenylalanineMetabolism in Perfused Rat Liver MOUSSA B. H. YOUDIM, B. MITCHELL and H. F. WOODS M.R.C. Clinical Pharmacology Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary, Oxford OX2 6HE, U.K. The pathways of phenylalanine metabolism in mammals have been widely invesigated, and the factors influencing phenylalanine hydroxylation in the liver have been elucidated by using purified enzyme preparations in vitro (see Kaufman, 1971, for a review). With the exception of the perfusion studies of Embden & Baldes (1913), there is little information about the metabolism of phenylalanine in the intact liver. Embden & Baldes (1913) demonstrated ketogenesis from phenylalanine in perfused dog liver. In the present communication we describe some aspects of phenylalanine metabolism in the isolated perfused rat liver. Livers from female Wistar rats (180-22Og) were perfused by using the method of Hems et al. (1966) with a semi-synthetic medium (Woods et al., 1970) which contained glucose (5mmol/litre) and phenylalanine (initial concn. 0-30mmol/litre). Perfusions continued for 2h, and samples of the medium were withdrawn for analysis at intervals. Isolated hepatocytes were prepared, and incubated by using the procedure described by Krebs et al. (1974). Perfusion of livers from rats fed with a basal medium alone resulted in the appearance of phenylalanine and tyrosine in the medium during the first 90min. The concentrations remained constant for the subsequent 30min. The final concentrations of these amino acids in the medium (0.06 mmol/litre for phenylalanine, and 0.04mmol/litre for tyrosine) arevery similar to thosereported by Schimassek & Gerok (1965) under similar conditions. These changes were accompanied by ketone-body (acetoacetate and 3-hydroxybutyrate) accumulation at a rate of 2.12 f 0.33pmol/h per g (s.E.M., four observations). When phenylalanine (1 mmol/litre) was present in the medium, there’was a removal of phenylalanineat an initial rate of 0.33 k 0.03pmol/minper ~(s.E.M., fiveobservations). This was accompanied by the appearance of tyrosine and ketone bodies in the medium, the removal of phenylalanine being fully accounted for by the accumulation of these products of the hydroxylation pathway. The rate of ketone-body production was 9.09 k 1.04 pmol/h per g (S.E.M., five observations). No fumarate or malate could be detected in the medium. The rate of the hydroxylation, as estimated by tyrosine and ketone-body
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BIOCHEMICAL SOCIETY TRANSACTIONS
production, increased with increasing initial phenylalanine concentration up to 5 mmol/ litre and then fell progressively. Although the rate of hydroxylation fell above this concentration, the initial rate of the phenylalanine removal increased over the same concentration range, being 0.6pmol/min per g at 5mmol/litre, and 1.5pmol/h per g at 30mmol/litre. These results suggest that at phenylalanine concentrations above 5 mmol/litre, alternative pathways of phenylalanine degradation are followed. Experiments in which 14C-labelledphenylalanine was incubated with isolated hepatocytes, and 14C02collected, showed that, at concentrations above 5 mmol/litre, there was a substantial decarboxylation of phenylalanine. The initial concentration of phenylalanine giving half-maximal rates of phenylalanine hydroxylation (‘apparent K,,,’) in the perfused liver was 1.50mmol/litre. This is similar to the K,,, for phenylalanine reported for purified rat liver phenylalanine hydroxylase assayed in the presence of dimethyltetrahydropterin[ 1.22-1.42mmol/litre (Gillam et al., 1974), 1.Ommol/litre (Kaufmann, 1969)l. In the perfused-liver experiment, no cofactor is added, thus the ‘apparent K,,,’ for phenylalanine is determined in the presence of the naturally occurring pterin cofactor present in the liver. In the presence of 1 mmol of phenylalanine/litre, the initial rate of phenylalanine hydroxylation, measured in the second hour of perfusion after the addition of a second load of phenylalanine (1 mmol/ litre) at 1h, is identical with that measured during the first hour. This suggests that the cofactor content of the liver is maintained over the 2h period. Intraperitoneal treatment of rats with p-chlorophenylalanine (300mg/kg for 2 days), a n inhibitor of phenylalanine hydroxylase, resulted in an almost complete inhibition of phenylalanine hydroxylation at phenylalanine concentrations below 5mmol/litre. However, under these conditions, phenylalanine removal still took place, suggesting that when the hydroxylation pathway is inhibited, phenylalanine is metabolized via alternative pathways under conditions where hydroxylation is normally the major reaction. Kaufmann (1971) has suggested that the inhibition of phenylalanine hydroxylase by its own substrate found in vitro is relevant to the regulation of the enzyme activity in vivo. The results presented above suggest that an alternative explanation of the decreased rate of hydroxylation at high concentrations of phenylalanine is the involvement of alternative reaction pathways having a K,,, for phenylalanine greater than that of phenylalanine hydroxylase. This explanation is also supported by the observation that at low phenylalanine concentrations, when the hydroxylase is inhibited by pchlorophenylalanine, phenylalanine metabolism still takes place. Ernbden, G. & Baldes, K. (1913) Biochem. Z . 55,301-322 Gillarn, S. S., Woo, S. L. & Woolf, L. I. (1974) Biochem. J. 139,731-739 Hems, R., Ross, B. D., Berry, M. N. & Krebs, H. A. (1966) Biochem. J. 101,284-292 Kaufrnan, S.(1969) Arch. Biochem. Biophys. 134,249-252 Kaufrnan, S . (1971) Ado. Enzymol. Relat. Areas Mol. Biol. 35,245-319 Krebs, H. A., Cornell, N. W., Lund, P. & Hems, R. (1974) Proc. Alfred Benzon Symp. 6th 726-750 Woods, H. F., Eggleston, L. V. & Krebs, H. A. (1970) Biochem. J. 119,501-510 Schirnassek,H. & Gerok, W. (1965) Biochem. Z . 343,407-415
The Metabolism of Some [l-’4C]Glycyl Dipeptides in Mice BARRY SAMPSON, BRIAN BARLOW and ANDREW WILKINSON Department of Paediatric Swgery, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K. Peptides occur as an important component (130%) of the total amino acid content of the protein hydrolysates used for intravenous feeding. The fate of peptides administered intravenously is largely unknown, although many workers have reported urinary losses of up to 40% of the peptide input (Christensen et al., 1947; Wei et al., 1972). 1975
683
i.e. oestrone sulphate, dehydroepiandrosterone sulphate, bromosulphophthalein and bilirubin, bound by aminoazodye-binding protein A, are also bound by ligandin, another carcinogen-binding protein widely distributed in the rat (Ketterer et al., 1967; Litwack et al., 1971). It is possible that these two binding proteins are functionally related. We thank the Cancer Research Campaign for a generous grant. Goodman, D. S. (1958) J. Am. Chem. SOC.80,3892-3898 Ketterer, B., Ross-Mansell, P. & Whitehead, J. K. (1967) Biochem. J. 103,316-324 Ketterer, B., Beale, D., Litwack, G. & Hackney, J. F. (1971) Chem.-Biol. Interact. 3,285-286 Litwack, G., Ketterer, B. & Arias, I. M. (1971) Nufure (London) 234,466-467 Mishkin, S . , Stein, L., Gatmaitan, 2. & Arias, I. M. (1972) Biochem. Biophys. Res. Commun. 47,997-1003
Mishkin, S. & Turcotte, R. (1974a) Biochem. Biophys. Res. Commun. 57,918-926 Mishkin, S . & Turcotte, R. (19746) Biochem. Biophys. Res. Commun. 60,376-381 Ockner, R. K., Manning, J. A., Poppenhausen,R. B. & Ho, W. K. L. (1972) Science 177.56-58 Rosenthal, H. E., Pietrzak, E., Slaunwhite, W. R. & Sandberg, A. A. (1972)J. Clin. Endocrinol. 34,805-81 3
Rudman, D., Bixler, T.J. & Del Rio, A. E. (1971) J. Phurmacol. Exp. Ther. 176,261-272
The Effect of Increasing Phenylalanine Concentration on PhenylalanineMetabolism in Perfused Rat Liver MOUSSA B. H. YOUDIM, B. MITCHELL and H. F. WOODS M.R.C. Clinical Pharmacology Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary, Oxford OX2 6HE, U.K. The pathways of phenylalanine metabolism in mammals have been widely invesigated, and the factors influencing phenylalanine hydroxylation in the liver have been elucidated by using purified enzyme preparations in vitro (see Kaufman, 1971, for a review). With the exception of the perfusion studies of Embden & Baldes (1913), there is little information about the metabolism of phenylalanine in the intact liver. Embden & Baldes (1913) demonstrated ketogenesis from phenylalanine in perfused dog liver. In the present communication we describe some aspects of phenylalanine metabolism in the isolated perfused rat liver. Livers from female Wistar rats (180-22Og) were perfused by using the method of Hems et al. (1966) with a semi-synthetic medium (Woods et al., 1970) which contained glucose (5mmol/litre) and phenylalanine (initial concn. 0-30mmol/litre). Perfusions continued for 2h, and samples of the medium were withdrawn for analysis at intervals. Isolated hepatocytes were prepared, and incubated by using the procedure described by Krebs et al. (1974). Perfusion of livers from rats fed with a basal medium alone resulted in the appearance of phenylalanine and tyrosine in the medium during the first 90min. The concentrations remained constant for the subsequent 30min. The final concentrations of these amino acids in the medium (0.06 mmol/litre for phenylalanine, and 0.04mmol/litre for tyrosine) arevery similar to thosereported by Schimassek & Gerok (1965) under similar conditions. These changes were accompanied by ketone-body (acetoacetate and 3-hydroxybutyrate) accumulation at a rate of 2.12 f 0.33pmol/h per g (s.E.M., four observations). When phenylalanine (1 mmol/litre) was present in the medium, there’was a removal of phenylalanineat an initial rate of 0.33 k 0.03pmol/minper ~(s.E.M., fiveobservations). This was accompanied by the appearance of tyrosine and ketone bodies in the medium, the removal of phenylalanine being fully accounted for by the accumulation of these products of the hydroxylation pathway. The rate of ketone-body production was 9.09 k 1.04 pmol/h per g (S.E.M., five observations). No fumarate or malate could be detected in the medium. The rate of the hydroxylation, as estimated by tyrosine and ketone-body
Vol. 3
684
BIOCHEMICAL SOCIETY TRANSACTIONS
production, increased with increasing initial phenylalanine concentration up to 5 mmol/ litre and then fell progressively. Although the rate of hydroxylation fell above this concentration, the initial rate of the phenylalanine removal increased over the same concentration range, being 0.6pmol/min per g at 5mmol/litre, and 1.5pmol/h per g at 30mmol/litre. These results suggest that at phenylalanine concentrations above 5 mmol/litre, alternative pathways of phenylalanine degradation are followed. Experiments in which 14C-labelledphenylalanine was incubated with isolated hepatocytes, and 14C02collected, showed that, at concentrations above 5 mmol/litre, there was a substantial decarboxylation of phenylalanine. The initial concentration of phenylalanine giving half-maximal rates of phenylalanine hydroxylation (‘apparent K,,,’) in the perfused liver was 1.50mmol/litre. This is similar to the K,,, for phenylalanine reported for purified rat liver phenylalanine hydroxylase assayed in the presence of dimethyltetrahydropterin[ 1.22-1.42mmol/litre (Gillam et al., 1974), 1.Ommol/litre (Kaufmann, 1969)l. In the perfused-liver experiment, no cofactor is added, thus the ‘apparent K,,,’ for phenylalanine is determined in the presence of the naturally occurring pterin cofactor present in the liver. In the presence of 1 mmol of phenylalanine/litre, the initial rate of phenylalanine hydroxylation, measured in the second hour of perfusion after the addition of a second load of phenylalanine (1 mmol/ litre) at 1h, is identical with that measured during the first hour. This suggests that the cofactor content of the liver is maintained over the 2h period. Intraperitoneal treatment of rats with p-chlorophenylalanine (300mg/kg for 2 days), a n inhibitor of phenylalanine hydroxylase, resulted in an almost complete inhibition of phenylalanine hydroxylation at phenylalanine concentrations below 5mmol/litre. However, under these conditions, phenylalanine removal still took place, suggesting that when the hydroxylation pathway is inhibited, phenylalanine is metabolized via alternative pathways under conditions where hydroxylation is normally the major reaction. Kaufmann (1971) has suggested that the inhibition of phenylalanine hydroxylase by its own substrate found in vitro is relevant to the regulation of the enzyme activity in vivo. The results presented above suggest that an alternative explanation of the decreased rate of hydroxylation at high concentrations of phenylalanine is the involvement of alternative reaction pathways having a K,,, for phenylalanine greater than that of phenylalanine hydroxylase. This explanation is also supported by the observation that at low phenylalanine concentrations, when the hydroxylase is inhibited by pchlorophenylalanine, phenylalanine metabolism still takes place. Ernbden, G. & Baldes, K. (1913) Biochem. Z . 55,301-322 Gillarn, S. S., Woo, S. L. & Woolf, L. I. (1974) Biochem. J. 139,731-739 Hems, R., Ross, B. D., Berry, M. N. & Krebs, H. A. (1966) Biochem. J. 101,284-292 Kaufrnan, S.(1969) Arch. Biochem. Biophys. 134,249-252 Kaufrnan, S . (1971) Ado. Enzymol. Relat. Areas Mol. Biol. 35,245-319 Krebs, H. A., Cornell, N. W., Lund, P. & Hems, R. (1974) Proc. Alfred Benzon Symp. 6th 726-750 Woods, H. F., Eggleston, L. V. & Krebs, H. A. (1970) Biochem. J. 119,501-510 Schirnassek,H. & Gerok, W. (1965) Biochem. Z . 343,407-415
The Metabolism of Some [l-’4C]Glycyl Dipeptides in Mice BARRY SAMPSON, BRIAN BARLOW and ANDREW WILKINSON Department of Paediatric Swgery, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K. Peptides occur as an important component (130%) of the total amino acid content of the protein hydrolysates used for intravenous feeding. The fate of peptides administered intravenously is largely unknown, although many workers have reported urinary losses of up to 40% of the peptide input (Christensen et al., 1947; Wei et al., 1972). 1975
