S63rd MEETING, LONDON
765
Further inspection of the ‘fingerprint’ (Fig. l), although strongly indicative of a globinlike protein, clearly did not suggest that it was a &chain globin, and eight out of the 24 major peptide spots found both chromatographed and stained identically with tryptic peptides expected from a digest of the embryonic ( E ) chain (Huehns e f al., 1964; Szelenyi & Hollan, 1969). This suggests that there may be some close similarity between the minor component observed in these thalassaemic patients and the &-chainfor which the amino acid sequence is not yet known. There was a complete absence of both trytophan and cysteine from both the E-chain and the polypeptide chain observed here. Clearly the results show that the minor component is a haem-protein complex, most probably a homotetramer, but until further work is carried out, it is premature to claim that it is in fact a true haemoglobin molecule. Baglioni, C. (1961) Biockini. Biophys. Acta 48, 392-396 Clegg, J. B., Naughton, M. A. & Weatherall, D. J. (1966)J. Mot. Biol. 19,91-108 Gray, W. R. (1967) Methods Enzyrnol. 11, 139-151 Huehns, E. R., Dance, N., Beaven, G. H., Keil, J. V., Hecht, F. & Motulsky, A. G. (1964)
Nature (London)201,1095-1097 Lang, A., Lehmann, H. & Ling-Lewis, P. A . (1974) Nature (London) 249 467469 Szelenyi, J. G. & Hollan, S. R. (1969) Acta Biochim. Biophys. Acad. Sci.Hung. 4,47-55 Weatherall, D. J. & Clegg, J. B. (eds.) (1972) The Thalassaerniu Syndrome, 2nd edn., Blackwell Science, London
Nucleotides and Human Placental Oestradiol-17P Dehydrogenase MICHAEL A. SHAW and JONATHAN JEFFERY Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 1AS, Scotland, U.K. The soluble fraction of human placenta contains 17/3- and 20a-hydroxy steroid dehydrogenase activities and reports suggest variously that the activities depend on the sameenzyme (Purdyet al., 1964; Jarabak &Sack, 1969)or that they do not (Karavolas & Engel, 1971). The possibility that the activities may be regulated by changes in the concentration of certain intracellular substances merits consideration; an effect of cyclic AMP, for example, might suggest that the regulation was linked to some other hormone or hormones, whereas an effect of ATP might suggest that regulation was linked to the metabolic state of the cell. A direct effect of prostaglandins has been excluded (Shaw & Jeffery, 1975). We now report studies of the effects of various nucleotides. The enzyme preparation was a stabilized solution made by homogenizing fresh placenta in suitable buffer, precipitating a broad fraction with ammonium sulphate, and partially purifying it by chromatography on DEAE-cellulose. The l7g-hydroxy steroid dehydrogenase activity was determined by using oestradiol-l7/l, oestrone, or 16ahydroxyoestrone as substrate, and following the change in the concentration of NADH or NADPH fluorimetrically (excitation at 337nm, emission at 465nm). In some cases the findings were confirmed by making absorbance measurements at 340nm. The 20ahydroxy steroid dehydrogenase activity was determined by using 20a-hydroxy-4pregnen-3-one as substrate and following the increase in the concentration of NADH or NADPH fluorimetrically (as described above). In the presence of 2 0 0 p ~ - N A D +as coenzyme, there was no marked effect of cyclic AMP (lOOfM-10m) on the rate of oxidation of oestradiol-178, nor of cyclic AMP (1 m-1 p ~ on ) the rate of oxidation of 20a-hydroxy-4-pregnen-3-one. The intracellular concentration of cyclic AMP in many tissues in vivo is thought to be 100nM-10pM, so the possibility that changes in these dehydrogenase activities might be mediated directly by cyclic AMP is excluded. Of 20 other nucleotides tested, most had little or no effect, but ATP inhibited the oxidation of both oestradiol-17fi and 20a-hydroxy-4-pregnen-3-one, with NAD+ a8
vol. 4
766
BIOCHEMICAL SOCIETY TRANSACTIONS
coenzyme. This inhibition was thought not to depend on covalent modification (e.g. a n ATP-dependent phosphorylation of the enzyme), because it had the following characteristics. It was effectiveas soon after mixing as measurements could be made, and was not increased by preincubation of the enzyme with ATP. It was relieved by dialysis, and was immediately restored by increasing the concentration of ATP again. It was competitive with respect to coenzyme insofar as the inhibition was greater at lower concentrations of coenzyme, and could be diminished by using high concentrations of coenzyme. When NAD+ (200pM) was used as coenzyme, the oxidation of oestradiol-178 (75pM) was inhibited by 83% by ~ O O ~ ~ M - A atTpH8 P in potassium phosphate buffer. The intracellular concentration of ATP in many tissues is of the order of 1mM; the concentration of NAD+ in human placenta is 2 0 0 , (Villee, ~~ 1962). Some inhibition by ATP may therefore occur in vivo. However, the effects of ATP may not be simple. There are certain differences in vitro according to whether NAD+, NADH, NADP+ or NADPH is the coenzyme used. Human placenta contains more NAD+ than NADH, and smaller amounts of NADPH and NADP+, of which the former predominates (Villee, 1962). Moreover, human placenta contains an enzyme (transhydrogenase) that catalyses the transfer of hydrogen between these coenzymes and for which oestradiol-178, but not oestrone, is an activator (Karavolas et al., 1969). We thank Professor H. M. Keir for his interest and encouragement, Mrs. Laura Selway for skilled technical assistance, and the Medical Research Council for a project grant. Jarabak, J. & Sack, G. H. (1969)Biochemistry 8,2203-2212 Karavolas, H.J. & Engel, L. L. (1971)Endocrinology88,1165-1169 Karavolas, H.J., Orr, J. C. & Engel, L. L. (1969)J.Biol. Chem. 244,4413-4421 Purdy, R.H.,Halla, M. & Little, B. (1964)Biochim. Biophys. Acta 89,557-560 Shaw, M.A.& Jeffery, J. (1975)Biochem. Soc. Trans. 3,889-890 Villee, C. A. (1962)l3iochem.J. 83, 191-194
Purification of Oestradiol Receptor by Chromatography on Oligo(dT)Cellulose C. HALL, S. THROWER, L. LIM and A. N. DAVISON Miriam Marks Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WCI N 3BG, U.K.
The action of oestradiol on the uterus is mediated by a specific receptor present in the cytosol. The receptor, after complexing with oestradiol, is translocated to the nucleus, where it influences transcription. In this step the 4 s cytoplasmic receptor complex is converted into a 5s ‘nuclear’ form (O’Malley & Means, 1974).DNA is an essential component in the nuclear binding (Shyamala, 1971) and DNA-cellulose has been used for the isolation of the oestradiol-receptor complex. Binding of the receptor compex to DNAcellulose mimics nuclear binding in vivo in that the receptor binds as the 5s form (Yamamoto & Alberts, 1972). Various other polynucleotides also bind the receptor (Sluyser et al., 1974). We have used column chromatography on oligo(dT) covalently bound to cellulose to isolate selectively the uterine oestradiol receptor in the 5s form. The technique is described in this communication, and in the following one (Thrower et al., 1976)we demonstrate that the conversion of the 4 s into the 5s form in vitro involve the participation of an essential activating factor. ‘Uterus cytosol’ is the 200000 g x 90min supernatant from a 25 % (w/v) homogenate of uteri isolated from Wistar female rats (60 days old) in ‘TED buffer’ (lOm-Tris/HCI /1 .Snm-EDTA/l mM-dithiothreitol, pH7.6). All experimental work was carried out at 4°C. Labelled oestradiol-receptor complex was produced by incubating cytosol with 5n~-[2,4,6,7-~H]oestradiol-178 (85Ci/mmol) at 4°C for 90min. Total receptor was determined as macromolecular-bound radioactivity excluded on Sephadex LH-20 1976
765
Further inspection of the ‘fingerprint’ (Fig. l), although strongly indicative of a globinlike protein, clearly did not suggest that it was a &chain globin, and eight out of the 24 major peptide spots found both chromatographed and stained identically with tryptic peptides expected from a digest of the embryonic ( E ) chain (Huehns e f al., 1964; Szelenyi & Hollan, 1969). This suggests that there may be some close similarity between the minor component observed in these thalassaemic patients and the &-chainfor which the amino acid sequence is not yet known. There was a complete absence of both trytophan and cysteine from both the E-chain and the polypeptide chain observed here. Clearly the results show that the minor component is a haem-protein complex, most probably a homotetramer, but until further work is carried out, it is premature to claim that it is in fact a true haemoglobin molecule. Baglioni, C. (1961) Biockini. Biophys. Acta 48, 392-396 Clegg, J. B., Naughton, M. A. & Weatherall, D. J. (1966)J. Mot. Biol. 19,91-108 Gray, W. R. (1967) Methods Enzyrnol. 11, 139-151 Huehns, E. R., Dance, N., Beaven, G. H., Keil, J. V., Hecht, F. & Motulsky, A. G. (1964)
Nature (London)201,1095-1097 Lang, A., Lehmann, H. & Ling-Lewis, P. A . (1974) Nature (London) 249 467469 Szelenyi, J. G. & Hollan, S. R. (1969) Acta Biochim. Biophys. Acad. Sci.Hung. 4,47-55 Weatherall, D. J. & Clegg, J. B. (eds.) (1972) The Thalassaerniu Syndrome, 2nd edn., Blackwell Science, London
Nucleotides and Human Placental Oestradiol-17P Dehydrogenase MICHAEL A. SHAW and JONATHAN JEFFERY Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 1AS, Scotland, U.K. The soluble fraction of human placenta contains 17/3- and 20a-hydroxy steroid dehydrogenase activities and reports suggest variously that the activities depend on the sameenzyme (Purdyet al., 1964; Jarabak &Sack, 1969)or that they do not (Karavolas & Engel, 1971). The possibility that the activities may be regulated by changes in the concentration of certain intracellular substances merits consideration; an effect of cyclic AMP, for example, might suggest that the regulation was linked to some other hormone or hormones, whereas an effect of ATP might suggest that regulation was linked to the metabolic state of the cell. A direct effect of prostaglandins has been excluded (Shaw & Jeffery, 1975). We now report studies of the effects of various nucleotides. The enzyme preparation was a stabilized solution made by homogenizing fresh placenta in suitable buffer, precipitating a broad fraction with ammonium sulphate, and partially purifying it by chromatography on DEAE-cellulose. The l7g-hydroxy steroid dehydrogenase activity was determined by using oestradiol-l7/l, oestrone, or 16ahydroxyoestrone as substrate, and following the change in the concentration of NADH or NADPH fluorimetrically (excitation at 337nm, emission at 465nm). In some cases the findings were confirmed by making absorbance measurements at 340nm. The 20ahydroxy steroid dehydrogenase activity was determined by using 20a-hydroxy-4pregnen-3-one as substrate and following the increase in the concentration of NADH or NADPH fluorimetrically (as described above). In the presence of 2 0 0 p ~ - N A D +as coenzyme, there was no marked effect of cyclic AMP (lOOfM-10m) on the rate of oxidation of oestradiol-178, nor of cyclic AMP (1 m-1 p ~ on ) the rate of oxidation of 20a-hydroxy-4-pregnen-3-one. The intracellular concentration of cyclic AMP in many tissues in vivo is thought to be 100nM-10pM, so the possibility that changes in these dehydrogenase activities might be mediated directly by cyclic AMP is excluded. Of 20 other nucleotides tested, most had little or no effect, but ATP inhibited the oxidation of both oestradiol-17fi and 20a-hydroxy-4-pregnen-3-one, with NAD+ a8
vol. 4
766
BIOCHEMICAL SOCIETY TRANSACTIONS
coenzyme. This inhibition was thought not to depend on covalent modification (e.g. a n ATP-dependent phosphorylation of the enzyme), because it had the following characteristics. It was effectiveas soon after mixing as measurements could be made, and was not increased by preincubation of the enzyme with ATP. It was relieved by dialysis, and was immediately restored by increasing the concentration of ATP again. It was competitive with respect to coenzyme insofar as the inhibition was greater at lower concentrations of coenzyme, and could be diminished by using high concentrations of coenzyme. When NAD+ (200pM) was used as coenzyme, the oxidation of oestradiol-178 (75pM) was inhibited by 83% by ~ O O ~ ~ M - A atTpH8 P in potassium phosphate buffer. The intracellular concentration of ATP in many tissues is of the order of 1mM; the concentration of NAD+ in human placenta is 2 0 0 , (Villee, ~~ 1962). Some inhibition by ATP may therefore occur in vivo. However, the effects of ATP may not be simple. There are certain differences in vitro according to whether NAD+, NADH, NADP+ or NADPH is the coenzyme used. Human placenta contains more NAD+ than NADH, and smaller amounts of NADPH and NADP+, of which the former predominates (Villee, 1962). Moreover, human placenta contains an enzyme (transhydrogenase) that catalyses the transfer of hydrogen between these coenzymes and for which oestradiol-178, but not oestrone, is an activator (Karavolas et al., 1969). We thank Professor H. M. Keir for his interest and encouragement, Mrs. Laura Selway for skilled technical assistance, and the Medical Research Council for a project grant. Jarabak, J. & Sack, G. H. (1969)Biochemistry 8,2203-2212 Karavolas, H.J. & Engel, L. L. (1971)Endocrinology88,1165-1169 Karavolas, H.J., Orr, J. C. & Engel, L. L. (1969)J.Biol. Chem. 244,4413-4421 Purdy, R.H.,Halla, M. & Little, B. (1964)Biochim. Biophys. Acta 89,557-560 Shaw, M.A.& Jeffery, J. (1975)Biochem. Soc. Trans. 3,889-890 Villee, C. A. (1962)l3iochem.J. 83, 191-194
Purification of Oestradiol Receptor by Chromatography on Oligo(dT)Cellulose C. HALL, S. THROWER, L. LIM and A. N. DAVISON Miriam Marks Department of Neurochemistry, Institute of Neurology, The National Hospital, Queen Square, London WCI N 3BG, U.K.
The action of oestradiol on the uterus is mediated by a specific receptor present in the cytosol. The receptor, after complexing with oestradiol, is translocated to the nucleus, where it influences transcription. In this step the 4 s cytoplasmic receptor complex is converted into a 5s ‘nuclear’ form (O’Malley & Means, 1974).DNA is an essential component in the nuclear binding (Shyamala, 1971) and DNA-cellulose has been used for the isolation of the oestradiol-receptor complex. Binding of the receptor compex to DNAcellulose mimics nuclear binding in vivo in that the receptor binds as the 5s form (Yamamoto & Alberts, 1972). Various other polynucleotides also bind the receptor (Sluyser et al., 1974). We have used column chromatography on oligo(dT) covalently bound to cellulose to isolate selectively the uterine oestradiol receptor in the 5s form. The technique is described in this communication, and in the following one (Thrower et al., 1976)we demonstrate that the conversion of the 4 s into the 5s form in vitro involve the participation of an essential activating factor. ‘Uterus cytosol’ is the 200000 g x 90min supernatant from a 25 % (w/v) homogenate of uteri isolated from Wistar female rats (60 days old) in ‘TED buffer’ (lOm-Tris/HCI /1 .Snm-EDTA/l mM-dithiothreitol, pH7.6). All experimental work was carried out at 4°C. Labelled oestradiol-receptor complex was produced by incubating cytosol with 5n~-[2,4,6,7-~H]oestradiol-178 (85Ci/mmol) at 4°C for 90min. Total receptor was determined as macromolecular-bound radioactivity excluded on Sephadex LH-20 1976
