Stimulation by oestradiol-17β of specific cytoplasmic and chromosomal protein synthesis in immature rat uterus

Molecular and Cellular Endocrinology

1 (1974) 21-36. @ North-Holland

Pub]. Comp.

STIMULATION BY OESTRADIOL-17P OF SPECIFIC CYTOPLASMIC AND CHROMOSOMAL PROTEIN SYNTHESIS IN IMMATURE RAT UTERUS R. J. B. KING*, Department

Dalia SOMJEN of Biodynamics,

**, A. M. KAYE

Weizmann

Institute

Received 16 November

and H. R. LINDNER

oj’science,

Rhovoth,

Israel

1973

1) We have studied the early induction by oestradiol-17S, of the synthesis of specific proteins in the cytosol and chromatin fractions of the immature (19-21 days) rat uterus. Surviving uteri from rats given 5 ug of oestradiol-17(3 by intraperitoneal injection were incubated for 1 h with 3H-leucine; uteri from control rats were incubated with 14C-leucine. Corresponding fractions were pooled, and the 3H/14C ratio in electrophoretically separated protein components was determined. 2) The oestradiol-17(3-induced protein (IP) originally described by Gorski and Notides (1969) was found to be localized in the lo5 g supernatant fraction of uteri homogenized in a medium which preserves the integrity of nuclei (0.25 M sucrose, 50 mM Tris buffer pH 7.5, 25 mM KC1 and 5 mM MgCh). No IP was found in liver from oestrogen-treated rats or in ovaries from rats treated with luteinizing hormone. 3) IP was found to have a molecular weight of 39,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and an isoelectric point of 4.5 as determined by isoelectric focusing in polyacrylamide gels. No evidence for phosphorylation of IP was obtained. 4) Extraction of uterine chromatin with 6 M urea, 0.4 M guanidinium chloride, 0.1 M sodium phosphate, pH 7.0, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated the presence of at least three oestradiol-induced proteins with molecular weights of 42,000,25,000 and 16,000 dalton respectively. The component of molecular weight 42,000 which is closest in size to IP was present in only small amounts. None of these components were ptesent in liver from oestrogen-treated rats. 5) Maximal amounts of the oestrogen-induced chromosomal proteins and of IP were found when the uteri were explanted 1 h after intraperitoneal injection of oestradiol-17S. 6) None of the oestradiol-induced proteins in uterine chromatin were histones, as judged from their molecular weight, but the component of molecular weight 16,000 was basic, as indicated by its adsorption to Bio-Rex-70 ion exchange resin. * This work was carried out whilst R.J.B.K. held an EMBO Fellowship. Present address to which reprint requests should be sent: Hormone Biochemistry Department, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WCZA 3PX, U.K. ** In partial fulfilment of the requirement for the Ph.D. degree of the Feinberg Graduate School of the Weizmann Institute of Science.

R. J. B. King et al.

22 Keywords: chromatin;

uterus; protein synthesis; induced protein; oestradioL17p

action.

The earliest increase in the synthesis of a uterine protein that has been observed, after injection of oestradiol-17B into immature or ovariectomized rats is that of a soluble protein which has been called induced protein (IP) (Mayo1 and Thayer, 1970; Gorski et al., 1971; Katzman et al., 1971). Recently, increased synthesis of the same protein component has been demonstrated in immature uteri incubated in the presence of oestradiol-17S (Katzenellenbogen and Gorski, 1972; Baulieu et al., 1972; Somjen et al., 1973a, b). Evidence quoted in the above papers has shown that this effect is steroid-specific and is dependent on de novo synthesis of protein and RNA. However, several important questions concerning IP have not yet been answered. Little is known about the location and properties of IP; for example, only one group (Baulieu et al., 1972) has reported data on its appearance in the nucleus. The appearance of oestrogeninduced nuclear proteins in the uterus has been reported (Teng and Hamilton, 1970; Barker, 1971). This paper describes various properties of nuclear and cytoplasmic oestradiol-17B-induced proteins and discusses their relationship to IP and to the proteins studied by Teng and Hamilton (1970) and Barker (197 1). Preliminary reports of parts of this work have been presented (Kaye et al., 1973; Sbmjen et al., 1973a).

MATERIALS

AND METHODS

Animals Wistar-derived female rats of the departmental colony were routinely used at the ages of 19 to 21 days; when lo-days old rats were used this is specifically stated. The maintenance of the rats has been described (Kaye et al., 1972). Materials Tritiated leucine (56-58 Ci/mmole), 14C-labelled leucine (330-342 mCi/ mmole) were obtained from the Radiochemical Centre, Amersham, U. K. and H 33‘PO4 (carrier free) was obtained from the Nuclear Research Center, Negev, Beer-Sheva, Israel, and H3 33P04 (carrier free) from New England Nuclear Corporation, Boston, Mass., U.S.A. y-labelled ATP was synthesized from these labelled phosphoric acid samples by the method of Glynn and Chappell(1964). Oestradiol-17B was purchased from Ikapharm Inc., Ramat-Gan, Israel, and ovine luteinizing hormone (NIH LH-S-17) was a gift of the Endocrine Section, U.S. National Institutes of Health, Bethesda, Maryland, U.S.A.

Stimulation

of protein

synthesis

by oestradiol-I

78

23

Bovine serum albumin, bovine pancreatic ribonuclease and beef heart cytochrome C were obtained from Sigma Chemical Co., St. Louis, MO., U.S.A. and ovalbumin and horse liver alcohol dehydrogenase from Worthington Biochemical Corp., Freehold, N. J., U.S.A. Ampholines were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden, Aquacide II and Miracloth from Calbiochem, Inc., Los Angeles, California, U.S.A. Cellogel (gelatinized cellulose acetate) strips, or 2.5 mm thick blocks from Chemetron, Inc., Milan, Italy, and Bio-Rex 70 ion exchange resin from Bio Rad Laboratories, Richmond, California, U.S.A. Chemicals for gel electrophoresis were obtained from Eastman Kodak Co., Rochester, N.Y., U.S.A. and chemicals for scintillation counting from Packard Instrument Co., Downers Grove, Illinois, U.S.A. Administration of hormones Oestradiol-17B solutions were prepared and injected as previously described (Kaye et al., 1972) at a dose of 5 ug/rat. Luteinizing hormone was dissolved in twice-distilled water and injected in a volume of 0.5 ml at a dose of 30 ug/rat. Controls received injections of 1% ethanol (oestradiol-17b vehicle) or twicedistilled water (LH vehicle). Incorporation qf amino acids into rat organs Three to 10 uteri or 10 pairs of ovaries were incubated in 1.65 ml of phosphate buffered saline (PBS) medium (Dulbecco and Vogt, 1954), control organs with 5 @/ml of 14C-leucine and organs from hormone treated rats with 25 &i/ml of 3H-leucine or with the isotopes reversed, i.e. controls with 14C-labelled and hormone-treated with 3H-labelled leucine. For induction of protein synthesis in vitro 3 uteri/ml were incubated in the presence of 3 x lo-’ M oestradiol-17j3 in PBS, for 1 h at 37 “C, and then transferred to PBS containing the appropriately labelled leucine. Incubations were done in 50 ml polypropylene centrifuge tubes under an atmosphere of O,-CO, (95.5 v/v). The tubes were agitated in a Dubnoff

shaker at about

one stroke per second.

Incorporation of 32P043- and 33P043Ten uteri were incubated in 1.5 ml of cell culture medium 199: control uteri in the presence of 150 pCi of 32P043- and uteri from oestradiol-17b-treated rats in 12.5 pCi of 33P043-. The supernatant solutions obtained by centrifugation of the uterine homogenates at 150,000 g were analysed, as were the fractions of the supernatants precipitated by (NH4)2S04 at 5&80 % saturation at 0 “C (Siimjen et al., 1973b).

24

R. J. B. King et al.

Fractionation of uteri After incubation, the organs were washed in PBS and then in TKM (50 mM Tris-HCl buffer pH 7.5 25 mM KCl, 5 mM MgCl,) medium (Blobel and Potter, 1966). Uteri (10) were homogenized in 0.25 M sucrose in TKM as previously described (Siimjen et al., 1973b). Crude nuclear-myofibrillar pellets were obtained by centrifugation at 700 g max for 10 min and were resuspended in 5 ml of 0.1 ‘A Triton X-100, 0.25 M sucrose in TKM and filtered through 8 layers of gauze and then through 2 layers of Miracloth. The filtrate was centrifuged at 700 gmax and the pellet was washed twice with 0.1 ‘A Triton X-100, 0.25 M sucrose in TKM and twice with 0.05 y0 disodium EDTA. The pellet was resuspended in 0.05 M Tris-HCl buffer, pH 8, and 0.5 ml of the suspension was layered on 3.0 ml of 1.7 M sucrose in 0.05 M Tris buffer pH 8.0 and centrifuged in the SW 56 rotor of a Beckman-Spinco ultra-centrifuge at 37,000 rpm (50,000 gaV) for 2 h. The resulting pellet was used as the chromatin fraction. The 700 gma, supernatant fraction was recentrifuged at 150,000 ga, for 50 min and the supernatant solution was considered the cytosol fraction. This fraction was kept at -20 “C, and centrifuged after thawing to remove a small amount of insoluble material. Storage for 2 months did not alter the content of IP. Extraction of nuclear proteins Nuclear proteins were extracted as described by Levy et al. (1972). The chromatin pellet was suspended (0.5 mg DNA/ml) in 6 M urea, 0.5 M guanidine hydrochloride, 0.1 M sodium phosphate pH 7.0 and 0.1% B-mercaptoethanol. The solution was stirred for 5 min and centrifuged overnight at 46,000 rev/min (200,000 g,,,) in the SW 56 rotor at 4 “C. The supernatant solution was called protein fraction 1. The pellet was resuspended in 0.2% sodium dodecyl sulphate (SDS), 0.1% P-mercaptoethanol, 0.01 M sodium phosphate buffer pH 7.0, dialyzed against this mixture for 24 h at room temp and then centrifuged as in the previous step, but at 20 “C. The resulting called protein fraction 2.

supernatant

solution

was

Extraction of cytoplasmic particulate fractions solution (see fractionation of In one experiment the 700 g,,, supernatant uteri) was centrifuged for 30 min at 10,000 g,,. The pellet was extracted with 0.5 ml 1 M NHICl for 10 min and then recentrifuged for 30 min at 10,000 g. The first 10,000 ga, supernatant solution from the above preparation was centrifuged at 100,000 g,, for 1 h. The pellet was extracted with 0.5 ml of 1 M NH.Cl for 10 min and recentrifuged at 100,000 g, for 1 h. Electrophoresis Cellogel Cellogel

electrophoresis

on 250 pm thick strips

in 0.04 M sodium

Stimulation of protein synthesis by oestradiol-17(3

25

barbitone pH 8.6 was carried out as described previously (Somjen et al., 1973b). Cellogel blocks (2.5 mm thick) were used at 14 v/cm for 2.5 h. For electrophoresis towards the cathode under acid conditions, the Cellogel strips (2.5 x 10 cm) were presoaked at least 10 min in 0.9 M acetic acid. After application of the sample (10-30 ul), electrophoresis was carried out in 0.9 M acetic acid at 2 mA per strip for 30 min. SDS-polyacrylamide gel Molecular weight determinations were made as described by Weber and Osborn (1969) using 4 x 75 mm gels. After application of the sample (100 ul), electrophoresis was initially at 5 mA/gel for 0.5 h; thereafter the current was increased to 8 mA/gel for a further 5 h. Crystalline bovine serum albumin (mol wt. 67,000) ovalbumin (2 times crystallized mol wt. 43,000), horse liver alcohol dehydrogenase (mol wt. 41,000), bovine pancreatic ribonuclease (5 times crystallized, mol wt. 13,700) and beef heart cytochrome C (mol wt. 11,700) were run simultaneously for calibration. Urea-polyacrylamide gel electrophoresis This was carried out as described by Panyim and Chalkley (1969), using 4 x 75 mm gels. After application of the sample (100 ul), initial electrophoresis was at 1 mA/gel for 1 h which was then increased to 2 mA/gel for a further 3-4 h. Isoelectricfocusing The gels were prepared by mixing 3 ml acrylamide solution (14.2 g acrylamide + 0.8 ml NN’-methylenebisacrylamide/lOO ml), 0.5 ml N,N,N’,N’ tetramethylene-diamine (1 % v/v), 1.45 ml 2 M sucrose, 0.8 ml glycerol, 0.6 ml ampholine pH 3-10, 1.15 ml HZ0 and lastly, 0.5 ml ammonium persulphate (5 mg/ml); 1.7 ml of this mixture was pipetted into 4 x 75 mm glass tubes and overlaid with water. After gelling, the tubes were transferred to a 4 “C cold room and left for at least 1 h before use. The gels were overlaid with 150 ul 5 % (w/v) sucrose containing 4 % (w/v) of the ampholine; 100 pl of sample in 7% (w/v) sucrose was pipetted underneath the 5 ‘A sucrose layer. The upper electrolyte solution (0.5% NaOH) was then added. The lower electrolyte solution was 0.02 % sulphuric acid. Electrophoresis was carried out in a 4 “C cold room at 1 mA/gel until a voltage of 400 V was reached then continued at 400 V. The total electrophoresis time was 10 h.

and

Scintillation counting Cellogel strips were cut into 2-mm sections, placed in vials containing 0.5 ml soluene and 5 ml scintillation solution and counted after at least 1 h at 2 “C. Polyacrylamide gels were cut into l-mm sections with a microtome type gel slicer and two sections were placed in each vial containing 0.5 ml soluene. The

26

R. J. B. King et al.

vials were capped and gently shaken at 37 “C overnight. Scintillation solution (5 ml) was then added and shaking was continued at room temp for a further 4-6 h. Samples were counted after standing in the dark at 2 “C for at least 1 h. SDS polyacrylamide and urea polyacrylamide gels were fixed in either 5 % (w/v) trichloroacetic acid or 10 % (w/v) sulphosalicylic acid for 30 min before slicing. To eliminate artefactual variation in the 3H/14C ratios in the isoelectric focusing gels, it was necessary to fix the gels in 5 ‘4 w/v trichloroacetic acid overnight before slicing. The scintillation solution contained 5 g 2,5-diphenyloxazole and 0.3 g dimethyl-l,3 bis (5 phenyloxazolyl-2)-benzene per litre toluene. Counting was done in a Packard Model 3310 scintillation spectrometer set so that less than 1 % 3H was counted in the 14C channel and 12-14 % 14C was counted in the 3H channel; a correlation was made for the crossover of i4C into the 3H channel. Quenching was found to be uniform for each isotope within each type of gel used. Separation of histone and non-h&one proteins

Bio-Rex 70 columns were used as described by Levy et al. (1972). After application of the sample to the column in 6 M urea, 0.4 M guanidinium chloride, 0.1 M potassium phosphate buffer pH 7.0, it was eluted at a rate of 10 ml/h with the same solution to obtain the non-basic protein fraction. Collecting fifteen fractions, each of 1.6 ml, the basic proteins were then removed with 6 M urea, 4 M guanidinium chloride, 0.1 M potassium phosphate buffer pH 7.0; these proteins were present in the 2nd and 3rd fractions after the buffer change. Concentration of samples

Protein solutions (0.2-1.0 ml) were concentrated in a dialysis bag immersed in Aquacide II. Protein determination

The method of Lowry et al. (1951) was used with bovine serum albumin as a standard.

RESULTS Intracellular distribution of cytoplasmic induced protein

Induced protein (IP) can be demonstrated in the high speed supernatant fractions obtained from uteri homogenized in sucrose-TKM (fig. la and b) or in EDTA (see refs. in Introduction) with equal efficiency. This makes it unlikely that the presence of IP in the cytoplasmic fraction is an artefact due to the use of EDTA. A component with the same electrophoretic mobility as

21

Stimulation of protein synthesb by oestradiol-I 78

60

a0 Migration

20

40

60

80

(mm)

Fig. 1. Cellogel analysis of oestradiol-17b induced cytoplasmic proteins. Rat organs were obtained 1 h after injection of 5 pg oestradiol-17b/rat (for curve(f) 30 pg luteinizing hormone/ rat) or of appropriate control solutions. In all cases except for curve (b) the control organs were incubated with ‘Gleu and the hormone treated organs with 3H-leu. Incubation, electrophoresis and cell fractionation were carried out as described in Materials and Methods. The arrows mark the positions of the origin and of bovine serum albumin respectively. (a) 150,000 gav supematant fraction from uterus; (b) similar extract except that the control uteri were incubated with 3H-Ieu and the oestradiol-17[3 stimulated uteri with %Xeu; (c) 1 M ammonium chloride extract from the 10,000 g,, pellet from uterine cytoplasm; (d) 1 M ammonium chloride extract from the 100,000 gaV pellet from uterine cytoplasm; (e) 150,000 g,, supematant fraction from liver; (f) 150,000 gav supematant fraction from ovary.

IP on Cellogel can be extracted from both the 10,000 ga, and the 100,000 g,, unwashed pellets of sucrose-TKM homogenates (fig. lc and d). It has been reported that a nuclear IP can be extracted with EDTA from a 15,000 g pellet (Baulieu et al., 1972). We found that radioactivity is extractable from such an unwashed pellet prepared according to the protocol of Baulieu et al. (1972) but that one wash with 0.1% Triton X-100 in 0.25 M sucrose removes all of the EDTA-extractable radioactive material. These results suggest that IP may be adsorbed non-specifically to particulate fractions. Oestradiol-17Bstimulated proteins associated with chromatin are described later in this paper. Spebjicity and time course of production of IP Cytoplasmic IP can be detected in uteri 1 h after injection

of 5 ug of oestrad-

28

R. J. B. King et al.

I

I

1

2 Tome after

I

3 oestradiolLl7p

I

4 (hr)

Fig. 2. Time course of induction by oestradioL17b of cytoplasmic protein from uterus. Rats were injected with either 5 ug oestradiol-17g or 0.5 ml of 1% ethanol. At the time shown, the uteri were removed, labelled with 3H- or W-leu analysed by Cellogel electrophoresis and the increase in IP measured by the Isotope ratio method of Katzenellenbogen and Gorski (1972). Results are expressed as the mean + range of 3-4 experiments.

iol-17p but not in liver from similarly treated rats (fig. le) nor in ovaries 1 h after luteinizing hormone injection (fig. If). In 20-day old rats more IP was detectable in uterine cytoplasm 1 h after injection of 5 ug oestradiol-17P than at 2 h and 4 h (fig. 2). However, in lo-day old rats IP showed the same level (approx. a 60% increase in the 3H/‘4C ratio) at 1 and 4 h after oestradiol-17l3 injection. Unless stated otherwise, all subsequent experiments on cytoplasmic protein induction were carried out with uteri from 19-21 day old rats 1 h after injection of 5 ug oestradiol-17lX Properties of cytoplasmic IP In 11 experiments the mean electrophoretic mobility of IP on Cellogel at pH 8.6 relative to that of bovine serum albumin was 1.11 with a range from 1.09-l .16. No peaks were detected when cytosol was electrophoresed in the opposite direction, towards the cathode, either at pH 8.6 or 3. Isoelectric focusing in polyacrylamide gels of cytoplasmic extracts indicated the presence of one major peak induced by oestradiol-17P (fig. 3). Three separate experiments gave a value for the isoelectric point of 4.5 i 0.1 (mean f SEM). A partial purification of cytoplasmic IP was possible by use of preparative scale Cellogel electrophoresis on thick gels (fig. 4a). The 2-cm

29

Stimulation of protein synthesis by oestradiol-l7fl

0’1.

‘.

\

‘\

8

‘\

7 ‘\ ‘\

*\

6 “1

20

c:

40 Mlgratfon

60

80

(mm)

Fig. 3. Analysis by isoelectric focusing in polyacrylamide gels of oestradiol-17fl-induced cytoplasmic proteins from uterus. Uteri were obtained 1 h after injection of either 5 pg oestradiol-17P/rat or 0.5 ml of 1% ethanol, incubated with 14C- or 3H-leu and subjected to isoelectric focusing as described in Materials and Methods.

region containing

the IP peak was recovered

and analysed

on a standard

thin

Cellogel strip (fig. 4b) showing that the bulk of the protein migrated in the IP region. Isoelectric focusing of such a partially purified IP showed an elevated 3H/14C ratio at pH 4.6 (fig. 4~). This indicates that the oestradioL17lGinduced peaks detected by Cellogel and isoelectric focu$ng are probably identical. SDS-acrylamide gel electrophoresis indicated that IP has a molecular weight of approx. 39,000 (table 1 and fig. 5). This size, together with its isoelectric point of 4.5 can adequately explain its electrophoretic mobility relative to bovine serum albumin on Cellogel and suggests that IP is not composed of subunits. Attempts were made to see if IP could be phosphorylated either by incubating uteri with radioactive inorganic orthophosphate or by incubating the high speed supernatant with radioactive ATP labelled in the y position. In

30

R. J. B. King et al.

0

20

40

60

Migration

(mm)

80

Fig. 4. Analysis of partly purified cytoplasmic induced protein from uterus by isoelectric focusing in polyacrylamide gels. Uteri were obtained from rats 1 h after injection of either 5 ug oestradiol-17b/rat or of 0.5 ml of 1% ethanol. Proteins were labelled by incubation of uteri with W- or 3H-leu as described in Materials and Methods. (a) radioelectropherogram on Cellogel blocks (2.5 mm thick) of uterine cytosol fractions. Material that migrated with the IP peak (*/cm) was expressed from the gel, using a 2-ml syringe, and subjected to electrophoresis on thin Cellogel strips (b), or to isoelectric focusing in polyacrylamide gels (c). The position of the induced protein is indicated by the SH/14C ratio curve.

these experiments, phosphorus3 3 was used for oestradiol-17P-treated uteri and phosphorus3’ for the control uteri. In both types of experiment, the labelled supernatants from control and oestradioL17l3 treated uteri were mixed and analysed both by Cellogel and polyacrylamide gel electrophoresis. Most of the phosphorylated components migrated more slowly than bovine serum albumin and no evidence was obtained for oestradiol-17P-induced phosphorylation in the IP region of the gels.

Stimulation of protein synthesis by oestradiol-17p

31

Table 1 Effect of oestradiol-17S treatment in vivo on the synthesis of some uterine proteins. Analysis by SDS-polyacrylamide electrophoresis. Animals were injected with either 0.5 ml of 1% ethanol or 5 ug oestradiol-17(3/rat. The uteri were removed at the times shown and processed as described in Materials and Methods. Peaks were detected by double isotope labelling and are defined in figs. 6 and 7. A minus sign indicates that the peak was not detected. Time after injection (h)

Molecular weight 1V Chromatin fraction 1 Peak no.

Expt. no.

1 2

4

Cytoplasmic

la

I-

2

3

1 2 3

62 54

_ 42 40

25 33 24

16 14 17

4 5 6 7

55 55 54 65

42 40 48 39

27 28 25 _

16 17 18

57 & 2

42 + 1

25 f 1

16 * 1

(Means & SEM)

23

40

60

80

100 Migration

59

I 29

I 14

I 75 lo-3

x

molecular

20

40

0 59

I 29

60

IP

42 38 38

39 rir 1

80

100

(mm) I 14

I 75

weiqht

Fig. 5. Comparison by SDS-polyacrylamide gel electrophoresis of proteins from uterine chromatin fraction 1 and cytoplasm. Uteri were obtained from rats 1 h after injection of either 5 ug oestradiol-17S/rat or of 0.5 ml of 1% ethanol. Proteins were labelled by incubation of uteri with W- or 3H-leu respectively. Incubations and preparation of chromatin (a, c) and cytosol fractions (b, d) are described in Materials and Methods. The arrows mark the position of the bromphenol blue marker. In (a) and (b), solid line, 3H radioactivity; broken line 14C radioactivity.

32

R. J. B. King et al.

Evidence for the presence of oestradio~--f7P_inducedproteins in trberinec~r~rn~~~~

Uterine chromatin prepared from detergent washed nuctei as described in Materials and Methods contains 3 or 4 oestradioI-17~-induced proteins that can be extracted with 6 M urea solution containing 0.4 M guan~dinium chloride

FGg. 6. S~S-polya~~I~~de gel elwtrophoresis of uterine chromatin protein fractions. 3 Pireparation of the fractions is described in Materials and Methods. (a> fraction 1, urea tlract; (b) fraction 2, SDS extract; (c) basic fraction from Bio-Rex 70 column; (d) non-b8 fraction from Ho-Rex 70 column.

33

Stimulation of protein synthesis by oestradiol-I 7(3

Migratton

(mm)

Fig. 7. SDS-polyacrylamide gel electrophoresis of fraction 1 proteins from chromatin. Organs were obtained from rats 1 h after injection of either 5 ug oestradiol-17S/rat or of 0.5 ml of 1% ethanol. (a) uterus; (b) liver. OestradioL17S treated preparations were incubated with 3H-leu and controls with 14C-leu. The arrows mark the position of the bromphenol blue marker. Solid line 3H: broken line W.

and 0.1 M sodium phosphate (figs. 5 and 7). A subsequent

buffer, pH 7.0 (chromatin fraction extraction with 0.2% (w/v) SDS,

1; (fig. 6)) 0.1% (v/v)

P-mercaptoethanol, 10 mM potassium phosphate buffer, pH 7.0, removed different proteins (fig. 6), but did not remove any different oestradiol-17g induced proteins regardless of whether the chromatins were isolated 1, 2 or 4 h after oestradiol-17P injection. Occasionally small amounts of material with the same electrophoretic mobility as the oestradiol-17P-induced proteins present in fraction 1 were detected in the SDS-extractable material but this fraction was not analysed further. Evidence that the material in fraction 1 is associated with the chromatin depends on the method used to purify the chromatin and, with the possible exception of a small amount of IP, the absence of similar peaks in the cytoplasm. Oestradiol-17P-induced proteins could be detected in fraction 1 regardless of which isotope (3H or 14C) was used for the treated and which for the control uteri. Since different amounts of leucine were used for 3H and 14C

34

R. J. B. King et al.

labelling, it is unlikely that the increased incorporation after oestradiol-17P treatment was due to changes in amino acid pool size. No preferentially labelled proteins were extractable from liver chromatin (fig. 7). Estimates of the molecular weights of the preferentially labelled proteins separated from uterine chromatin, 1, 2 and 4 h after intraperitoneal injection of oestradiol-17P are listed in table 1. The peaks with the largest change in 3H: 14C ratio were 2 and 3. These had apparent moleculer weights of 25,000 and 16,000 respectively. Peak 1 whose molecular weight (approx. 42,000) is closest to that of cytoplasmic IP, was a minor component. In 5 out of 9 experiments, evidence for its presence rested on a change in the 3H: i4C ratio in a single gel fraction. Peak la (mol wt. approx. 57,000) was not detected in 3 of 10 experiments. In 3 experiments molecular weight estimates were not obtained and hence they are not shown in table 1. Overnight dialysis of cytoplasmic IP against the urea-containing medium used to extract fraction 1 neither altered the subsequent mobility of IP on SDS gel nor resulted in the appearance of new peaks. It is therefore unlikely that the nuclear peaks are extraction artefacts of material analogous to the supernatant IP. The peak 3 material was retained by Bio-Rex 70 but no further data is available about the properties of these nuclear proteins.

DISCUSSION The presence of IP in the high speed supernatant from uteri homogenized in sucrose-TKM indicates that it is a cytosol component. While it is detected in unwashed mitochondrial and microsomal pellets, its ease of removal by ammonium chloride suggests the presence of IP in these particulate fractions may be an adsorption artefact. The decreased amounts of IP detected 2 and 4 h after oestradiol-17P treatment confirms precious data (Mayo1 and Thayer, 1970; Gorski et al., 1971; De Angelo and Fujimoto, 1973) for rats of approximately 20 days of age. The lack of such a decrease in lo-day old rats provides a contrasting system for further study of the translational control of IP synthesis proposed by De Angelo and Fujimoto (1973). The absence of IP in oestradiol17gtreated liver and in luteinizing hormone-treated ovary extends previously published data (see Introduction) on the specificity of IP production. Our results that IP is a protein of molecular weight approx. 39,000 and isoelectric point of pH 4.5 could account for its electrophoretic mobility in Cellogel, starch gels or acrylamide gels if it were not composed of subunits. Gorski and Notides (1969) have reported that IP migrates as a single anionic

35

Stimulation of protein synthesis by oestradiol-I 7p

peak between pH 5.9 and 9.0 on starch gels and have further data that the isoelectric point is below pH 5.0 (Gorski, pers. commun.). Mayo1 and Thayer (1970) have stated that multiple oestradiol-17P-induced uterine proteins focus between pH 3.540 on pH 3-5 gradients. Our data indicate that uterine chromatin contains at least three proteins whose synthesis is stimulated by oestradiol-17B. These are found after in vivo administration of oestradiol-17P. None appear to be histones as judged from their electrophoretic mobility, although material in peak 3 with a molecular weight of approx. 16,000 behaves as a basic protein on Bio-Rex 70 ion exchange columns. A relationship between these nuclear components and the cytoplasmic IP is not established. As judged from the SDS gels, the molecular weight of nuclear component 1 is close to that of IP but the amount of this chromatin component is small. We cannot at present exclude the possibility that IP may be located in a non-chromatin fraction of the nucleus. Further work is necessary to see whether the other oestradiol-17P-induced protein components of the chromatin are separate, unrelated entities or whether any of them are formed from IP during a hypothetical passage into the nucleus. Baulieu et al. (1972) have reported that IP can be extracted by EDTA from a 15,000 g uterine pellet. Our inability to extract significant radioactivity from Triton-washed nuclei with EDTA supports the view that the IP studied by these workers was not associated with chromatin. Teng and Hamilton (1968) have detected by 3H tryptophan labelling, elevated amounts of a NaOH extractable nuclear protein in uterus, but not liver, 12 h after injecting oestradiol-17p into rats. Comparison of their data with ours is complicated by the different extraction regimes used, but it is noteworthy that their component migrated on SDS-urea polyacrylamide gels as relatively low molecular weight material. Barker (1971) studying the incorporation of a 14C-amino acid mixture by the uterus in vivo has reported the presence of an oestradiol-17P-induced acid-soluble chromatin protein, larger than the histones. Further work is required to establish the relationship of our data to that of Barker’s. It is interesting that IP has a similar molecular weight to the non-histone protein induced in liver by cortisol (Shelton and Allfrey, 1970) and to the ecdysone-induced non-histone protein from salivary glands of Drosophila (Helmsing, 1972).

ACKNOWLEDGEMENTS This work

was supported

by grants

from

the Ford

Foundation

and the

36

R. J. B. King et al.

Population Council, New York, U.S.A. A.M.K. is the Herbert Sidebotham Senior Research Fellow and H.R.L. the Adlai E. Stevenson Professor of Endocrinology and Reproductive Biology at the Weizmann Institute of Science.

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