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Binding of thyrotropin to adiposscell membranes from the guinea pig was rapid under standard incubation conditions, and half-maximal association occurred within 1min. A decrease in the temperature of incubation from 37°C to 4°C led to a decrease in the rate of association. At 37"C, longer incubation periods ( 2 4 h ) resulted in a slight decrease in binding, whereas binding at lower temperatures appeared stable. Preincubation studies indicated that this effect was due primarily to instability of the membranes. Binding of 1251-1abelledthyrotropin to adipose-cell membranes was decreased with increasing concentrations of unlabelled thyrotropin, and the binding sites appeared saturable. Conventional Scatchard analysis suggests the presence of a high-affinity receptor, with an affinity constant of the order of 4 x l O 9 ~ - lsimilar , to that ascribed to thyroid membranes (Manley et al., 1974; Tate et al., 1975). Significant competition for thyrotropin-binding sites was not observed when excess amounts of lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), insulin, somatotropin (growth hormone), adrenaline or noradrenaline were added, confirming the specificnature of the binding. Binding of 1251-labelledthyrotropin was inhibited by concentrations of NaCl or KCI above that already included in the assay buffer (0.03 M), and specific binding was abolished when the concentration of either ion was 0 . 5 or ~ above. Both CaC12 and MgClz enhanced specific binding when present at 3 mM, higher concentrations being inhibitory. Propranolol (1 mM) also increased specific binding and in a similar manner to its reported effect on thyroid membranes (Marshall et al., 1976), which is presumed to be a function of the local-anaesthetic properties of this drug. Binding of bovine thyrotropin to adipose-cell membranes has been detected; it is rapid, saturable and specific and of similar affinity and stability to that seen with thyroid membranes. D. L. G. is grateful for support from a Medical Research Council training award, and thanks are due to Dr. J. G. Pierce for the supply of bovine thyrotropin. Bray, G. A. & Trygstad, 0. (1972) Acfa Endocrinol. 70, 1-20 Goldfine, I. D., Amir, S. M.. Ingbar, S. H. & Tucker, G. (1976) Biochim. Biophys. Acra 448, 45-56 Hart, I. R. & McKenzie, J. M. (1971) Endocrinology 88, 26-30 Karonen, S. L., Morsky, P., Siren, M. & Seuderling, U. (1975) Anal. Biochem. 67, 1-10 Lowry, 0.H., Rosebrough,N.J.,Farr, A. L. &Randall, R. J. (1951)J.Biol. Chem. 193,265-275 Manley, S . W., Bourke, J. R. & Hawker, R. W. (1974) J. Endocrinol.61,419436 Marshall, N. J., Florin-Christensen,A. & von Borcke, S. (1976)Biochem. Soc. Trans.4,149-151 Mehdi, S. Q.& Nussey, S. S. (1975) Biochem. J. 145, 105-111 Mullin, B. R., Lee, G., Ledley, F. D., Winand, R. J. & Kohn, L. D. (1976) Biochem. Biophys. Res. Commun. 69,55-62 Tate, R. L., Schwartz, H. I., Holmes, J. M., Kohn, L. D. & Winand, R. J. (1975)J. Biol. Chem. 250,6509-6515 Teng, C. S., Smith, B., Anderson, J. & Hall, R. (1976) Biochem. Biophys. Res. Commun. 66, 836-841

Alkylation of Methionine Residues in Pituitary Growth Hormone OLIVIA GASTON-PARRY and MICHAEL WALLIS School of Biological Sciences, University of Sussex, Falmer, Brighton B N 1 9QG, Sussex, U.K.

Methionine residues in proteins can be modified by alkylation with iodoacetic acid or iodoacetamide at low pH (Gundlach el al., 1959). We have previously used alkylation with iodoacetic acid to prepare a derivative of bovine growth hormone (somatotropin) in which the methionine residues were carboxymethylated (Wallis, 1972). This derivative

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BIOCHEMICAL SOCIETY TRANSACTIONS

Table I . Reuctioities oj’methioirine residues iir hoiliire growth hormone Bovine growth hormone was alkylated with the ’‘C-labelled alkylating agent shown, in glycine/HCl buffer, pH3.5 (0.1 M with respect to glycine). Sp. radioactivity of methionine residue (d.p.m./nmol) Alkylating agent Iodoacetic acid Iodoacet amide

c

Met-5 5900 982

5

Met-124 1475 1910

Met-149 18500 4560

Met-179 12100 3680

was virtually inactive in a growth-promoting assay. In addition, alkylation with this reagent was used to investigate the relative reactivities of the four methionine residues in growth hormone. In the present communication we describe further studies on the alkylation of bovine growth hormone, using both iodoacetic acid and iodoacetamide as alkylating agents. Modification of human growth hormone has also been investigated. The procedure used to study the reactivity of the methionine residues to alkylation was to treat the hormone with a small quantity of the 14C-labelledalkylating reagent (iodoacetic acid or iodoacetamide: 0.5 mol/mol of methionine residue) for 24h. Under these conditions the methionine side chains compete for the alkylating agent, the most reactive residues being most heavily labelled. The hormone was subsequently treated with an excess of unlabelled alkylating agent, in the presence of a denaturing agent ( 8 ~ - u r e aor 5 M-guanidinium chloride), to complete alkylation of the methionine side chains. The completely modified product was then subjected to trypsin digestion (followed by chymotryptic digestion for the bovine hormone) and the resulting peptide mixture was fractionated by paper chromatography and electrophoresis. Radioactive peptide spots were detected by radioautography, eluted and analysed for amino acid content (after acid hydrolysis) and radioactivity. The specific radioactivity of each peptide gave a measure of the reactivity of the (single) methionine residue that it contained. When this procedure was used for the carboxymethylation of bovine growth hormone, the results obtained previously (Wallis, 1972) were confirmed. In a wide range of buffers, at pH3.5 and 2.0, it was found that the two methionine residues nearest the C-terminus (Met-149 and Met-179) were most reactive, Met-5 (near the N-terminus) was rather less reactive, and Met-124 was much less reactive than the other three. A typical result is shown in Table 1. When iodoacetamide was used as the alkylating agent, giving carboxyamidomethylated growth hormone, Met-149 and Met-179 were again most reactive, but in this case Met-124 was rather more reactive than Met-5 (Table 1). Again, the same pattern of labelling could be repeated in a wide range of buffers and at pH3.5 and 2.0. In all the studies, it was found that methionine residues were the only ones labelled to a significant extent, as expected at these low pH values. Preliminary alkylation experiments have also been carried out with human growth hormone. This has only three methionine residues and has an amino acid sequence that differs considerably from that of the bovine hormone. The results show that Met-125 has relatively low reactivity compared with Met-14 or Met-170. It is noteworthy that the methionine at residue 125 (residue 124 in bovine growth hormone) is relatively unreactive in growth hormone from both species, and is the only one that is conserved in the two sequences (and is indeed conserved in all the other growth-hormone sequences that have been determined, as well as in the related human placental lactogen; Wallis, 1975). In another series of experiments we have investigated the biological activity of growth hormone modified by alkylation of methionine residues. The alkylation was carried out with excess of unlabelled iodoacetic acid or iodoacetamide, in the presence 1977

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of urea as denaturant. This ensured complete modification of methionine residues. The growth-promoting activity of the derivatives was determined in an assay using hereditary dwarf mice (Wallis & Dew, 1973). Bovine growth hormone which had been carboxyamidomethylated on all four methionine residues showed a substantial loss of activity in the growth-promoting assay; no activity was detected at the highest dose tested (40pg/day). The lack of activity is thus similar to that observed previously for the carboxymethylated hormone (Wallis, 1972). However, human growth hormone that had been carboxyamidornethylated on its three rnethionine residues did retain slight activity in the dwarf-mouse assay.

We thank Mr. Peter Dew for carrying out the amino acid analyses, Mrs. Molly Reed and Mr. George Blank for expert management of the dwarf-mouse colony and the Medical Research Council for financial support. Gundlach, H. G., Moore, S. &Stein, W. H. (1959)J. Biol. Chem.234,1761-1764 Wallis, M. (1972) FEBS Lett. 21, 118-122 Wallis, M. (1975) Biol. Rev. Cambridge Philos. SOC. 50, 35-98 Wallis, M. & Dew, J. A. (1973) J. Endocrinol. 56, 235-243

Hypothalamic and Peripheral Relations during the Oestrous Cycle of the Female Rat: the Oestrogen Receptor in the Uterus SEAN THROWER, LOUIS LIM* and JOHN 0. WHITE Miriam Marks Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London W C l N 3BG, U.K.

Variations in the plasma concentration of oestradiol play a key role in the regulation and maintenance of the oestrous cycle in rats (Brown-Grant et al., 1970). This variationis the result of the regulated release of gonadotropins from the anterior pituitary, which in turnis under the tropic influence of the hypothalamus (Schwartz & McCormack, 1972). There are oestradiol-binding sites in pre-optic hypothalamic regions of the brain (McEwen et al., 1974) and we have begun investigations on the role of the oestrogen receptor in the regulation of gonadotropin release. We have investigated the relationship of the cytosol and nuclear oestrogen receptors by simultaneous measurements of oestradiol binding in the uterus and the hypothalamus (White et al., 1977). There are contradictory reports on the behaviour of the oestrogen receptor in the uterus during the cycle (see Schwartz & McCormack, 1972; Brenner & West, 1975), although changes in the amount of receptor in the nucleus appear to follow the pattern of plasma oestradiol concentration (Clark et al., 1972). We confirm the nuclear pattern and show that cytosol receptor concentration is maintained throughout the cycle. Groups of 42 female Wistar rats, purchased from Charles River U.K. Ltd., were kept in our animal house (12 h dark/l2 h light schedule) for a week before use. Vaginal smears were examined on several successive days including the day of experiment, to monitor progress through the oestrous cycle. Some95 % of therats followed a4-day cycle: oestrus and pro-oestrus lasted 1 day each; the other 2 days are described here as late dioestrus and metoestrus/early dioestrus. Rats were killed at mid-day by decapitation, and the uteri, brain and liver removed. Uteri werepooledaccordingto phase, chopped finely, and homogenized in 3 vol. of buffer (10mM-Tris/HC1, pH7.6, 1m ~ - E D T A , 1mM-dithiothreitol), containing 5 n ~ - [ ~ H ] oestradiol. Nuclear and cytosol fractions were prepared, and the high-affinity macromolecular binding of [3H]oestradiol was determined for each fraction by (respectively) the technique of Anderson et al. (1972) and exclusion chromatography on Sephadex

* To whom reprint requests should be addressed. VOl. 5