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(Siiteri &Wilson, 1970)and in vitro (Giorgi et al., 1971), it should be possible to compare the relative radioactivity in the tissue and perfusate with the corresponding data for dihydrotestosterone to get an indication of the avidity of the tissue for each metabolite. Thus %reduced metabolites with at least one hydroxyl group at either C-3 or C-17B are retained by the tissue, possibly more avidly than dihydrotestosterone, whereas the diketones are apparently transferred back into the medium more readily than is dihydrotestosterone. Bruchovsky, N. & Wilson, J. D. (1968)J. Biol. Chem. 243,2012-2021 Giorgi, E. P., Stewart, J. C., Grant, J. K. &Scott, R. (1971) Biochem. J. 123,41-55 Giorgi, E. P., Moses, T. F., Grant, J. K., Scott, R. & Sinclair, J. (1974) Mol. Cell Endocrinol. 1,271-284

Harper, M. E. Pierrepoint, C. G., Fahmy, A. R. & Grifiths, K. (1971) J . Endocrinol. 49,213223

King, R. J. B. & Mainwaring, W. I. P. (1974) Steroid Cell Interactions, pp. 50-54, Butterworth, London Robel, P. (1971) Acta Endocrinol. (Copenhagen)Suppl. 153,279-294 Siiteri, P. K. &Wilson, J. D. (1970)J. Clin. Invest. 49,1737-1745 Wilson, J. D. (1972) N . Eng1.J. Med. 287,1284-1291 Wilson, J. D. & Gloyna, R. E. (1970) Recent Prog. Horm. Res. 26,309-329

Interactions of Steroid Sulphates, Long-Chain Fatty Acids and Related Compounds with a Low-Molecular-WeightCarcinogen-BindingProtein from Rat Liver EDWARD TIPPING, BRIAN KETTERER, LUCIA CHRISTODOULIDES and GRAHAM ENDERBY Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London W lP 5PR, U.K. Aminoazodye-binding protein A was first isolated by Ketterer et al. (1967). The protein has a molecular weight of 14000 determined by gel filtration, and can be resolved by isoelectric focusing into three components with PI values of 5.4 (component I), 6.1 (component 11) and 7.4 (component 111), each with the same amino acid composition (Ketterer et al., 1971). Refocusing of single components results in redistribution to all three forms but there is a marked tendency towards the formation of component 111. Each purified component binds non-covalently a wide range of substances including steroid sulphates, bilirubin, bromosulphophthalein, palmitate, oleate and palmitoylCoA. These binding properties and the molecular weight suggest that aminoazodyebinding protein A is identical with Z protein (Mishkin et al., 1972)and fatty acid-binding protein (Ockner et al., 1972) found in rat intestinal mucosa, liver, myocardium, adipose tissue, skeletal muscle and kidney. To date the only quantitative binding studies have been made by gel filtration with partially purified protein (Mishkin et al., 1972; Mishkin & Turcotte, 1974~).The present communication reports equilibrium-dialysis measurements of the binding of severalcompounds to purified and partially purified aminoazodyebinding protein A. Potassium [6,7-3H]oestrone sulphate (specific radioactivities 480 and 5800 Ci/mol), potassium dehydr0[7-~H]epiandrosterone sulphate (4600Ci/mol), disodium bromo[35S]sulphophthalein (25.3 Ci/mol), [9,10-3H]palmitic acid (500 Ci/mol) and [9,103H]oleic acid (2500 Ci/mol) were purchased from The Radiochemical Centre, Amersham, Bucks., U.K. [l-14C]Palmitoyl-CoA was from NEN Chemicals G.m.b.H., Frankfurt, Germany. Where necessary radioactive oestrone sulphate and dehydroepiandrosterone sulphate were diluted with unlabelled materials [Sigma (London) Chemical Co., Kingston-upon-Thames, Surrey, U.K.]. Radioactivity was counted in a Packard TriCarb 3375 liquid-scintillation spectrometer. Quench correction was made by the external-standard method. 1975

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Azodye-free aminoazodye-binding protein A was purified as described previously (Ketterer et al., 1967), except that a final step involving zone electrophoresiswas replaced by isoelectric focusing on an LKB 8100 column containing Ampholines of pH range 3.5-10 (LKB Produkter AB, Stockholm, Sweden) and a discontinuous 0 4 0 % sucrose density gradient. After focusing (48-72 h) aminoazodye-binding protein A was identified in fractions eluted from the column by its characteristic u.v.-absorption spectrum which displays considerable fine structure between 250 and 270nm and a distinct shoulder at 285nm. The binding of [6,7-3H]oestrone sulphate to each fraction was measured by equilibrium dialysis in specially constructed PTFE cells. These enabled 0.25ml of protein-plus-ligand solution (made by adding 0.1 ml of each fraction to 0.7ml of 0 . 3 , ~ ~ oestrone sulphate solution) to be dialysed against 0.25ml of buffer. Membranes were cut from 18/32 Visking dialysis tubing. Measurements were made in 0.1 M-KC14.025MK2HP04 buffer, adjusted to pH7.0 with H3P04 at 4 (+l)”C. The results are shown in Fig. 1.

1.0111

0

.

j

0.6 -

-_

8

1

q“ 0.4

-

0.2 -

Fraction number Fig. 1. Binding of oestrone subhate by components I, II, and ZIZ of aminoazodye-binding protein A 0 , EzB0;----, pH; 0, oestrone sulphate binding to individual fractions measured by equilibrium dialysis (for details see the text). Results are expressed as fraction of oestrone sulphate bound.

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Table 1. Binding parameters for aminoazodye-bindingprotein A The association constant and the number of binding sites are given for the purified protein. The binding capacity is expressed as mol of ligand bound/g of l00OOOg supernatant protein. Purified aminoazodye-binding Aminoazodye-binding protein A protein A fraction , ? Ligand 107xBinding n log K capacity log K Oestrone sulphate Dehydroepiandrosterone sulphate Bromosulphophthalein Palmitate Oleate Palmit oyl-CoA

5.5

1

5.2 5.4

1 2

5.0* 5.0*

-

6.3

1

-

6.4 6.3 6.4

1.2

5.2

1.1 2.8 0.8

5.6t 7.8, 6.6$

1.7

-

* IognK values. t Result of Mishkin et al. (1972).

$ Result of Mishkin & Turcotte (1974~).

After purification component I11 was always present in the largest amount and was the strongest binder for all ligands studied. The significance of forms I and I1 is not clear; they may be due to tight binding of a charged ligand, or to differences in protein conformation. In this study association constants and numbers of binding sites were determined for form I11 only. Equilibrium-dialysis measurements were carried out at protein concentrations of 5 - 2 0 ~ ~Similar . experiments were performed on ‘aminoazodye-binding protein A fraction’, the fraction corresponding to a molecular-weight range of 1OOOO18000 after gel filtration on Sephadex G-100 of the l00OOOg supernatant from rat liver. The results are shown in Table 1. Aminoazodye-binding protein A fraction bound all ligands studied more strongly (6-30-fold) than did purified protein. This might be explained by the observation that, after dialysis of lOml of aminoazodye-binding protein A fraction against 10 litres of KCI-phosphate buffer, the binding affinities for oestrone sulphate and dehydroepiandrosterone sulphate were decreased 10-20-fold, suggesting that the strong binding of these two ligands at least by aminoazodye-binding protein A requires a diffusible molecule. This loss of high affinity would also occur in the purification procedure. Non-radioactive oestrone sulphate at a concentration of 0.1 mM (an excess of 100-fold or more) did not affect the binding of palmitoyl-CoA to aminoazodye-binding protein A fraction, suggesting more than one kind of binding site. Inview of this it is possible that the protein is involved in more than one physiological process. One function of the protein seems to be the stimulation of microsomal acyl-CoAsn-glycerol 3-phosphate acyltransferase (Mishkin & Turcotte, 19746). Another possibility is that the protein is part of an ‘intracellular sink’ involved in the passive uptake of substances from blood to liver and other tissues. In the case of free fatty acids this would seem unlikely in view of the high affinity of serum albumin for these compounds (Goodman, 1958) and the relatively low affinity of aminoazodye-binding protein A. However, oestrone sulphate and dehydroepisandrosterone sulphate (Rosenthal et al., 1972) and bromosulphophthalein (Rudman et al. ,1971) are bound less tightly by albumin than by aminoazodye-binding protein A, and a role in uptake of these compounds cannot be ruled out. A third possible function is regulation of intracellular concentrations of free ligands. In connexion with the last two functions it should be noted that several compounds, 1975

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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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