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Electrophoretic separation of prolactin secreted from mouse pituitary glands in vitro
313
Molecular and Cellular Endocrinology, 33 (1983) 313-320 Elsevier Scientific Publishers Ireland. Ltd. MCE 01084
ELECTROPHORETIC SEPARATION OF PROLACTIN FROM MOUSE PITUITARY GLANDS IN VITRO H. DOHI, S. SAKAI, K. KOHMOTO Department
SECRETED
and Y. SHODA
of Animal Breeding, University of Tokyo, Bunkyo-ky
Tokyo 113 (Japan)
Received 6 June 1983; accepted 22 September 1983
Prolactin secreted from the mouse pituitary gland in organ culture was characterized after disc or SD~polyac~Iamide gel electrophoresis. Some pituitary glands were cultured with ‘H-labelled leucine for 24 h to obtain radioactive prolactin. In disc electrophoresis, immunoreactive, receptor-bindable and ‘H-incorporated prolactins formed a single band with the same relative mobility (0.5). Prolactin migrating at R, = 0.5 was extracted and re-analysed by SDS electrophoresis. A single stained and radioactive band was observed at the same position with an apparent molecular weight of 23000 regardless of denaturing in the presence of absence of di~othreitol. No other band was detected. These results indicate that mouse prolactin synthesized and secreted in organ culture is homogeneous and that mouse prolactin is a single-chain molecule. Keywords:
immunoreactivity;
receptor binding; organ culture; prolactin.
Rat and mouse PRLs are a single polypeptide chain with a molecular weight of about 23000, as shown by the Ferguson’s plot and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Cheever and Lewis, 1969; Cheever et al., 1969; Colosi et al., 1982) and amino acid sequence studies (Parlow and Shame, 1976; K. Kohmoto, unpublished). A single-chain peptide product was synthesized in a cell-free translation assay directed by mRNA for rat PRL (Evans and Rosenfeld, 1976; Maure et al., 1976; Evans et al., 1977), However, heterogeneous PRLs which have larger mobilities than predominant PRL were shown electrophoretically. Sex, age, strain and physiological differences in the animals are associated with the production of heterogeneous PRL (Asawaro~ngchai et al., 1978; Sinha and Baxter, 1979a; Sinha, 1980). Mittra (1980) found that PRL has a cleavage in its large disulphide loop revealed by SDS-PAGE in the rat pituitary and in the culture medium. Pituitary glands cultured in a synthetic medium secrete a large amount of PRL and a small amount of GH for many days with smaller amounts 0303-7207/83/$03.~
@ 1983 Elsevier Scientific Publishers Ireland, Ltd.
314
H. Dohi et al.
of other proteins. In these experiments, PRL secreted from pituitary glands was analysed by polyacrylamide gel disc electrophoresis (discPAGE), since GH has a different mobility on the gel and is separated easily from PRL. In order to express the PRL action, PRL binds to the PRL receptor of target organs (i.e. mammary gland) in the same species. We examined first the position of radioimmunoassayable, receptor-bindable and radioactive PRL on the gel after disc-PAGE. Even if PRL separated by the present method is thought to be considerably pure, it is also possible that the PRL band may contain certain amounts of non-PRL proteins (Zanini et al. 1974). The heterogeneity of PRL and other proteins present in the PRL band was further assessed by SDS-PAGE with or without dithiothreitol. We observed that the PRL band contains only the PRL molecule with an entire single peptide chain. MATERIALS
AND METHODS
Materials L-[3H]leucine and NCS tissue solubilizer were purchased from the Radiochemical Centre (Amersham, U.K.); SDS molecular weight standard from Sigma Chemical Co. (St. Louis, MO, U.S.A.); Medium 199, Waymouth’s MB752/1 Medium and MEM Alpha Medium (a-MEM) from Grand Island Biological Co. (Grand Island, NY, U.S.A.); insulin from Novo Industri A/S (Bagsvaerd, Denmark); ovine PRL (NIH-P-S12) was supplied by NIAMDD. Chemicals used for electrophoresis were from Wako Pure Chemical Industries (Osaka, Japan). Organ culture of pituitary glands Pituitary glands from 3-month-old virgin KA mice were aseptically removed. Three glands were cultured in 0.5 ml media ((u-MEM, Waymouth’s MB752/1 or Medium 199) supplemented with 0.12 IU/ml insulin on a floating paper raft at 37°C in moistened gases of 95% 0, and 5% CO, for 10 days. Culture media were collected every 24 h and stored at - 20°C until use. In order to prepare radioactive PRL, pituitaries were cultured in a-MEM with 5 pCi[3H]leucine for 24 h before the termination of culture. Polyacrylamide gel disc electrophoresis Culture media were subjected to electrophoresis with a discontinuous buffer system (disc-PAGE, Ornstein and Davis, 1969) on 10.5% acrylamide gels. After electrophoresis, gels were cut into 1 mm thickness and protein from each gel segment was eluted in 100 ~1 10 mM phosphate buffer (pH 6.9)-0.25% bovine serum albumin (BSA) at 4°C overnight. Amounts of PRL in these eluates were measured by radioimmunoassays
Secreted mouse prolactin
315
(RIA) and radioreceptor assays (RRA). In electrophoresis of 3H-labelled PRL, the gel was washed with successive changes of 7% acetic acid after staining. Protein in each gel segment was solubilized with 0.3 ml NCS tissue solubilizer at 50°C for 3 h. The radioactivity was measured in 6 ml scintillation mixture. The position of radioimmunoassayable, receptorbindable and 3H-labelled PRL was always compared to that of bands in Amido Black-stained gels. Medium samples obtained from 1 to 10 days of culture were also electrophoresed on a slab gel plate. The position and the density of stained bands were monitored using a densitometer (Nichion Ika-Rika Seisakusho, Funabashi, Japan). SDS-polyacrylamide gel disc electrophoresis The PRL band was eluted from a gel in 200 ~1 10 mM Tris-HCl (pH 6.7) containing 5 pg BSA and 5 pg ovalbumin overnight at 4°C precipitated with 10% trichloroacetic acid (TCA), and centrifuged at 1000 x g for 5 min. The pelleted sample was dissolved in 10 ~1 1 N NaOH and 80 ~1 sample buffer containing 1% SDS-10 mM Tris-HCl (pH 6.8)-5.6 M urea with or without 100 mM dithiothreitol, and denatured for 2 min in a boiling water bath. The denatured protein was electrophoresed on a 12.5% polyacrylamide gel in the presence of 0.2% SDS (SDS-PAGE) according to the method of Laemmli (1970). The gel was fixed with 25% methanol-7% acetic acid and stained with 0.25% Coomassie Blue in the same fixative. For molecular weight estimation, BSA (67 000) ovalbumin (45 000), chymotrypsinogen A (25 000), myoglobin (17 800) and cytochrome c (12 400) were used as standards. Radioimmunoassay and radioreceptor assay of mouse PRL Methods for RIA and RRA have been described elsewhere (Harigaya et al., 1982; Sakai et al., 1978). A homologous RIA using the double antibody technique showed a dose-response relationship for 0.1-l ng purified mouse PRL. Samples diluted serially from 1 : 50 to 1 : 800 with 0.15 M NaCl + 1% BSA + 10 mM phosphate buffer (pH 7.5) were used for the assay. The amount of PRL was expressed in terms of purified mouse PRL on a weight basis. The RRA system consisted of collagenase-dissociated lactating mouse epithelial cells (400 ~1; 4 x lo6 cells), enzymatically iodinated ovine PRL (90 yl; spec. act. 80 pCi/pg, 1.5 ng), and unlabelled mouse PRL (10 pl; 12.5-800 ng, 5 pg) or unknown samples (10 ~1; 1: 0 or 1: 1 with Medium 199 + 0.25 BSA). The reaction was allowed to continue for 2 h at room temperature with mild agitation (50 cycles/mm). At the end of the reaction, the mammary cells were sedimented by 5-10 set centrifugation in an Eppendorf microfuge and washed twice in 1 ml of Hank’s balanced salt solution containing 0.25% BSA. Radioactivity in the pelleted mammary cells was measured in
316
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an auto-gamma counter. Specific binding of 1251-labelled PRL was obtained by subtraction of radioactivity in the presence of 5 pg ovine PRL from that in the absence. A dose-related inhibition of ‘251-labelled ovine PRL was obtained from 12.5 to 800 ng unlabelled mouse PRL. This displacement curve was parallel to that obtained for the serial dilution of pituitary, medium or eluted samples. Inter- and intra-assay variations were approximately 10.2% and 15.5% respectively, from 10 experiments. RESULTS The ability of mouse PRL to compete with ‘25f-labelled ovine PRL for receptor sites on mouse m~mary cells was examined by RRA (Fig. 1). Purified mouse PRL inhibited ‘2sI-labelled PRL binding to the receptor
x4x2x1 Dilution
I
I
I
I
IO
100
Hormone concentrotlon
( ng 1
IO{
oh 0
Distance from
2
3 origin
( cm)
Fig. 1. Dose-response curves for ovine prolactin (m) and mouse prolactin (e) in a radioreceptor assay employing ‘251-labelled ovine prolactin as tracer and mouse mammary cells as a source of receptor. The pituitary homogenate (Sl, 0) and the medium sample obtained from l-day culture (SZ, r) were electrophoresed by disc-PAGE. The prolactin band (1 mm thickness) was cut and &ted in 100 plO.15 M NaCl-15 mM phosphate buffer (pH 6.9) at 4OC overnight. The sample was diluted to 1: 1, 1: 2 and 1: 4 with the above solution. 10 pl of each sample was used for a radioreceptor assay. Fig. 2. Radioactivity distribution on disc-PAGE gel, After pituitaries had been cultured in ol-MEM with [3H]leucine for 24 h, the medium was subjected to disc-PAGE. Each 1 mm segment from a stained, washed gel was solubilized for radioactivity counting. Electrophoretogram of proteins stained with Amide Black 10B is shown at the top.
4
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in a dose-related manner but was a less effective competitor than ovine PRL. The serial dilution of pituitary or secreted PRL eluted from gels after PAGE showed the inhibition of ‘251-labelIed ovine PRL binding to be in parallel with the dose vs. response curve for purified mouse PRL. When the pituitary culture was subjected to disc-PAGE, two distinct bands (GH, R, = 0.25, and PRL, R, = 0.50) were always detected on gels stained with Amido Black 10B through the lo-day period of culture in cu-MEM and Waymouth’s medium. The PRL band was stained consistently the darkest among the protein bands separated. No other bands were detected, except for a very faint band which was observed just in front of the PRL band when gels were stained with Coomassie Blue. These bands were diffused in gels when pituit~ies were cultured in Medium 199. When pituitaries were cultured with i3H]leucine in (u-MEM and the medium was electrophoresed, the major peak of radioactivity was seen at R, = 0.50, where the stained PRL band was present (Fig. 2). The GH band was less radioactive. The distributions of radioimmunoassayable mouse PRL and of receptor-binding ability on gels are shown in Fig. 3. Both these activities are observed only at R, = 0.50, coinciding exactly with the mobility of mouse PRL on the gel. Protein which reacts with the
0
I 2 Distance from origin
( cm I
3
Fig. 3. Distribution of the radioimmunoassayable and receptor-bindable PRL on disc-PAGE gel. 4- or 5-day cultured pituitaries were cultured for 24 h and the medium was electrophoresed. PRL activities in eluates from sliced gels were determined by RIA (e) and RRA (0).
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Fig. 4. SDS-PAGE of PRL denatured with (A) and without (B) dithiothreitol. PRL and [3H]PRL were eluted from PRL bands which were separated by disc-PAGE. After electrophoresis in SDS, gels were stained with Coomassie Blue and the radioactivity distribution was determined. Arrows indicate BSA and ovalbumin which were added to eluates as carrier proteins for TCA precipitation. For the culture conditions see Fig. 2.
anti-mouse PRL serum and the mammary PRL receptor site could not be detected in other places on the gel through the lo-day period of culture. The eluted PRL band was denatured in the presence of SDS with or without dithiothreitol and was analysed again by SDS-PAGE. Figs. 4A and 4B show the PRL band stained with Coomassie Blue and the radioactivity distribution of 3H-incorporated protein on the gel. In each figure, stained band and radioactivity were separated as a single band with the same mobility. The molecular weight of mouse PRL was estimated to be 23000 on SDS gels regardless of denaturing in the presence or absence of dithiothreitol. However, PRL and molecular weight marker proteins denatured in the absence of dithiothreitol migrated much faster and diffused more than those denatured in the presence of dithiothreitol.
DISCUSSION Proteins in the medium were separated into two bands, faintly and darkly stained with Arnido Black lOB, and had relative mob&ties of 0.25 and 0.5, respectively, on polyacrylamide gels. Only these two bands were labelled with [3H]leucine. A higher radioactivity was found in the PRL band, and a lower activity in the GH band. Of these two, only the PRL band reacted to anti-mouse PRL serum and also displaced [‘251]ovine
Secreted mouse proiacrin
319
PRL from mouse mammary PRL receptors. The mobility of PRL agrees with those observed in tissue homogenates (Cheever et al., 1969; Lewis et al., 1969). When longer gels were used and stained with Coomassie Blue, one minor band ~grating faster than PRL was found. This charge isomer is probably the deamidated form of mouse PRL (Lewis et al., 1970; Kohmoto, 1975; Farmer et al., 1976). PRL bioactivities, determined by Pigeon crop-sac assays (Cheever et al., 1969; Kohmoto and Bern, 1971; Yanai and Nagasawa, 1974) and by the mouse mammary organ culture (Nicholson, 1969), are principally associated with the PRL band on polyacrylamide gels. Size and charge heterogeneities of PRL have been reported in mice (Sinha and Baxter, 1979a, b) and rats (Dombroske et al., 1976; Aswaroengchai et al., 1978; Wallis et al., 1980). However, the present data show that the PRL receptor-binding activity and immunoreactivity were present at the position of the most darkly stained and heavily 3H-labelled band. Mouse PRL denatured with or without dithiothreitol was separated as a single band with an estimated molecular weight of 23000, as reported by Shore et al. (1978) and Colosi et al. (1982). Mittra (1980) observed intact and cleaved PRL in the culture medium of rat pituitaries on SIX-PAGE. This cleavage of PRL may take place after translation because only a single-chain prePRL was synthesized in a cell-free translation directed by mRNA for rat PRL (Evans et al., 1976). No cleaved form of mouse PRL similar to Mittra’s fraction was observed in our mouse pituitary organ culture system. In analysis of rat and bovine pituitaries or of the medium of bovine pituitary slices cultured in vitro, protein bands equivalent to cleaved PRL could not be found (Zanini and Rosa, 1981). If such cleaved PRL does not bind to either anti-mouse PRL serum or mammary PRL receptors and migrate with a different mobility, these PRLs may be excluded for the subsequent SDS-PAGE in these experiments. However, we observed only two protein bands (GH and PRL) in the stained gel. Cultured pituitaries secrete a large amount of homogeneous PRL into the medium through 10 days. The secreted PRL definitely cross-reacts with anti-mouse PRL serum, binds to mouse mamm~y PRL receptors and migrates with a single mobility both on disc and SDS gels. We conclude, therefore, that the secreted PRL is a single-chain molecule having a molecular weight of 23 000.
ACKNOWLEDGEMENTS We are grateful to NIAMDD
for the supply of ovine PRL (NIH-P-
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Sl2), to Mr. T. Kondo for taking care of animals, and to Mrs. K.imiko Tachikawa for typing the manuscript.
REFERENCES Asawaroengchai, H., Russel, SM. and Nicoll, C.S. (1978) Endocrinology 102, 407-414. Cheever, E.V. and Lewis, U.J. (1969) Endocrinology 85,465-473. Cheever, E.V., Seavey, B.K. and Lewis, U.J. (1969) Endocrinology 85, 698-703. Colosi, P., Marr, G., Lopez, J., Ogren, L. and Talamantes, F. (1982) Proc. Nat]. Acad. Sci. (U.S.A.) 79, 771-775. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. Dombroske, O.C., Payne, P. and Fran@ W.L. (1976) Fed. Proc. 35, 219 (abstract). Evans, G.A. and Rosenfeld, M.G. (1976) 3. Biol. Chem. 215, 2842-2847. Evans, G.A., Hucko, J. and Rosenfeld, M.G. (1977) Endocrinology 101, 1807-1814. Farmer, S.W., Bewley, T.A., Russel, SM. and Nicoll, C.S. (1976) Biochim. Biophys. Acta 437, 562-570. Harigaya, T., Sakai, S., Kohmoto, K. and Shoda, Y. (1982) J. Endocrinol. 94, 149-155. Kohmoto, K. (1975) Endocrinol. Jpn. 22, 465-469. Kohmoto, K. and Bern, H.A. (1971) Proc. Sot. Exp. Biol. Med. 137, 807-809. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Lewis, U.J., Littreia, M. and Cheever, E.V. (1969) Endocrinology 85, 690-697. Lewis, U.J., Cheever, E.V. and Hopkins, W.C. (1970) Biochim. Biophys. Acta 214,498-508. Maure, R.A., Stone, R. and Gorski, J. (1976) J. Biol. Chem. 251, 2801-2807. Mittra, I. (1980) B&hem. Biophys. Res. Commun. 95, 1750-1759. Nicholson, P.M. (1969) J. Endocrinol. 44, 459-460. Omstein, 0. (1964) Ann. N.Y. Acad. Sci. 121, 321-349. Parlow, A.F. and Shome, B. (1976) Fed. Proc. 35, 219. Sakai, S., Enami, J., Nandi, S. and Banerjee, M.R. (1978) Mol. Cell. Endocrinol. 12, 285-298. Shore, L.F., Shine, N.R. and Talamantes, F. (1978) Biochim. Biophys. Acta 537, 336-347. Sinha, Y.N. (1980) Endocrinology 107, 1959-1969. Sinha, Y.N. and Baxter, S.R. (1979a) Biochem. Biophys. Res. Commun. 86, 325-330. Sinha, Y.N. and Baxter, S.R. (1979b) J. Endocrinol. 81, 229-314. Wallis, M,, Daniels, M. and Ellis, S.A. (1980) Biochem. J. 189, 605-614. Yanai, R. and Nagasawa, H. (1974) J. Endocrinol. 62, 685-686. Zanini, A. and Rosa, P. (1981) Mol. Cell. EndocrinoI. 24, 165-179. Zanini, A., Giannattasio, G. and Meldolesi, J. (1974) Endocrinology 94, 594-598.
Molecular and Cellular Endocrinology, 33 (1983) 313-320 Elsevier Scientific Publishers Ireland. Ltd. MCE 01084
ELECTROPHORETIC SEPARATION OF PROLACTIN FROM MOUSE PITUITARY GLANDS IN VITRO H. DOHI, S. SAKAI, K. KOHMOTO Department
SECRETED
and Y. SHODA
of Animal Breeding, University of Tokyo, Bunkyo-ky
Tokyo 113 (Japan)
Received 6 June 1983; accepted 22 September 1983
Prolactin secreted from the mouse pituitary gland in organ culture was characterized after disc or SD~polyac~Iamide gel electrophoresis. Some pituitary glands were cultured with ‘H-labelled leucine for 24 h to obtain radioactive prolactin. In disc electrophoresis, immunoreactive, receptor-bindable and ‘H-incorporated prolactins formed a single band with the same relative mobility (0.5). Prolactin migrating at R, = 0.5 was extracted and re-analysed by SDS electrophoresis. A single stained and radioactive band was observed at the same position with an apparent molecular weight of 23000 regardless of denaturing in the presence of absence of di~othreitol. No other band was detected. These results indicate that mouse prolactin synthesized and secreted in organ culture is homogeneous and that mouse prolactin is a single-chain molecule. Keywords:
immunoreactivity;
receptor binding; organ culture; prolactin.
Rat and mouse PRLs are a single polypeptide chain with a molecular weight of about 23000, as shown by the Ferguson’s plot and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Cheever and Lewis, 1969; Cheever et al., 1969; Colosi et al., 1982) and amino acid sequence studies (Parlow and Shame, 1976; K. Kohmoto, unpublished). A single-chain peptide product was synthesized in a cell-free translation assay directed by mRNA for rat PRL (Evans and Rosenfeld, 1976; Maure et al., 1976; Evans et al., 1977), However, heterogeneous PRLs which have larger mobilities than predominant PRL were shown electrophoretically. Sex, age, strain and physiological differences in the animals are associated with the production of heterogeneous PRL (Asawaro~ngchai et al., 1978; Sinha and Baxter, 1979a; Sinha, 1980). Mittra (1980) found that PRL has a cleavage in its large disulphide loop revealed by SDS-PAGE in the rat pituitary and in the culture medium. Pituitary glands cultured in a synthetic medium secrete a large amount of PRL and a small amount of GH for many days with smaller amounts 0303-7207/83/$03.~
@ 1983 Elsevier Scientific Publishers Ireland, Ltd.
314
H. Dohi et al.
of other proteins. In these experiments, PRL secreted from pituitary glands was analysed by polyacrylamide gel disc electrophoresis (discPAGE), since GH has a different mobility on the gel and is separated easily from PRL. In order to express the PRL action, PRL binds to the PRL receptor of target organs (i.e. mammary gland) in the same species. We examined first the position of radioimmunoassayable, receptor-bindable and radioactive PRL on the gel after disc-PAGE. Even if PRL separated by the present method is thought to be considerably pure, it is also possible that the PRL band may contain certain amounts of non-PRL proteins (Zanini et al. 1974). The heterogeneity of PRL and other proteins present in the PRL band was further assessed by SDS-PAGE with or without dithiothreitol. We observed that the PRL band contains only the PRL molecule with an entire single peptide chain. MATERIALS
AND METHODS
Materials L-[3H]leucine and NCS tissue solubilizer were purchased from the Radiochemical Centre (Amersham, U.K.); SDS molecular weight standard from Sigma Chemical Co. (St. Louis, MO, U.S.A.); Medium 199, Waymouth’s MB752/1 Medium and MEM Alpha Medium (a-MEM) from Grand Island Biological Co. (Grand Island, NY, U.S.A.); insulin from Novo Industri A/S (Bagsvaerd, Denmark); ovine PRL (NIH-P-S12) was supplied by NIAMDD. Chemicals used for electrophoresis were from Wako Pure Chemical Industries (Osaka, Japan). Organ culture of pituitary glands Pituitary glands from 3-month-old virgin KA mice were aseptically removed. Three glands were cultured in 0.5 ml media ((u-MEM, Waymouth’s MB752/1 or Medium 199) supplemented with 0.12 IU/ml insulin on a floating paper raft at 37°C in moistened gases of 95% 0, and 5% CO, for 10 days. Culture media were collected every 24 h and stored at - 20°C until use. In order to prepare radioactive PRL, pituitaries were cultured in a-MEM with 5 pCi[3H]leucine for 24 h before the termination of culture. Polyacrylamide gel disc electrophoresis Culture media were subjected to electrophoresis with a discontinuous buffer system (disc-PAGE, Ornstein and Davis, 1969) on 10.5% acrylamide gels. After electrophoresis, gels were cut into 1 mm thickness and protein from each gel segment was eluted in 100 ~1 10 mM phosphate buffer (pH 6.9)-0.25% bovine serum albumin (BSA) at 4°C overnight. Amounts of PRL in these eluates were measured by radioimmunoassays
Secreted mouse prolactin
315
(RIA) and radioreceptor assays (RRA). In electrophoresis of 3H-labelled PRL, the gel was washed with successive changes of 7% acetic acid after staining. Protein in each gel segment was solubilized with 0.3 ml NCS tissue solubilizer at 50°C for 3 h. The radioactivity was measured in 6 ml scintillation mixture. The position of radioimmunoassayable, receptorbindable and 3H-labelled PRL was always compared to that of bands in Amido Black-stained gels. Medium samples obtained from 1 to 10 days of culture were also electrophoresed on a slab gel plate. The position and the density of stained bands were monitored using a densitometer (Nichion Ika-Rika Seisakusho, Funabashi, Japan). SDS-polyacrylamide gel disc electrophoresis The PRL band was eluted from a gel in 200 ~1 10 mM Tris-HCl (pH 6.7) containing 5 pg BSA and 5 pg ovalbumin overnight at 4°C precipitated with 10% trichloroacetic acid (TCA), and centrifuged at 1000 x g for 5 min. The pelleted sample was dissolved in 10 ~1 1 N NaOH and 80 ~1 sample buffer containing 1% SDS-10 mM Tris-HCl (pH 6.8)-5.6 M urea with or without 100 mM dithiothreitol, and denatured for 2 min in a boiling water bath. The denatured protein was electrophoresed on a 12.5% polyacrylamide gel in the presence of 0.2% SDS (SDS-PAGE) according to the method of Laemmli (1970). The gel was fixed with 25% methanol-7% acetic acid and stained with 0.25% Coomassie Blue in the same fixative. For molecular weight estimation, BSA (67 000) ovalbumin (45 000), chymotrypsinogen A (25 000), myoglobin (17 800) and cytochrome c (12 400) were used as standards. Radioimmunoassay and radioreceptor assay of mouse PRL Methods for RIA and RRA have been described elsewhere (Harigaya et al., 1982; Sakai et al., 1978). A homologous RIA using the double antibody technique showed a dose-response relationship for 0.1-l ng purified mouse PRL. Samples diluted serially from 1 : 50 to 1 : 800 with 0.15 M NaCl + 1% BSA + 10 mM phosphate buffer (pH 7.5) were used for the assay. The amount of PRL was expressed in terms of purified mouse PRL on a weight basis. The RRA system consisted of collagenase-dissociated lactating mouse epithelial cells (400 ~1; 4 x lo6 cells), enzymatically iodinated ovine PRL (90 yl; spec. act. 80 pCi/pg, 1.5 ng), and unlabelled mouse PRL (10 pl; 12.5-800 ng, 5 pg) or unknown samples (10 ~1; 1: 0 or 1: 1 with Medium 199 + 0.25 BSA). The reaction was allowed to continue for 2 h at room temperature with mild agitation (50 cycles/mm). At the end of the reaction, the mammary cells were sedimented by 5-10 set centrifugation in an Eppendorf microfuge and washed twice in 1 ml of Hank’s balanced salt solution containing 0.25% BSA. Radioactivity in the pelleted mammary cells was measured in
316
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an auto-gamma counter. Specific binding of 1251-labelled PRL was obtained by subtraction of radioactivity in the presence of 5 pg ovine PRL from that in the absence. A dose-related inhibition of ‘251-labelled ovine PRL was obtained from 12.5 to 800 ng unlabelled mouse PRL. This displacement curve was parallel to that obtained for the serial dilution of pituitary, medium or eluted samples. Inter- and intra-assay variations were approximately 10.2% and 15.5% respectively, from 10 experiments. RESULTS The ability of mouse PRL to compete with ‘25f-labelled ovine PRL for receptor sites on mouse m~mary cells was examined by RRA (Fig. 1). Purified mouse PRL inhibited ‘2sI-labelled PRL binding to the receptor
x4x2x1 Dilution
I
I
I
I
IO
100
Hormone concentrotlon
( ng 1
IO{
oh 0
Distance from
2
3 origin
( cm)
Fig. 1. Dose-response curves for ovine prolactin (m) and mouse prolactin (e) in a radioreceptor assay employing ‘251-labelled ovine prolactin as tracer and mouse mammary cells as a source of receptor. The pituitary homogenate (Sl, 0) and the medium sample obtained from l-day culture (SZ, r) were electrophoresed by disc-PAGE. The prolactin band (1 mm thickness) was cut and &ted in 100 plO.15 M NaCl-15 mM phosphate buffer (pH 6.9) at 4OC overnight. The sample was diluted to 1: 1, 1: 2 and 1: 4 with the above solution. 10 pl of each sample was used for a radioreceptor assay. Fig. 2. Radioactivity distribution on disc-PAGE gel, After pituitaries had been cultured in ol-MEM with [3H]leucine for 24 h, the medium was subjected to disc-PAGE. Each 1 mm segment from a stained, washed gel was solubilized for radioactivity counting. Electrophoretogram of proteins stained with Amide Black 10B is shown at the top.
4
317
in a dose-related manner but was a less effective competitor than ovine PRL. The serial dilution of pituitary or secreted PRL eluted from gels after PAGE showed the inhibition of ‘251-labelIed ovine PRL binding to be in parallel with the dose vs. response curve for purified mouse PRL. When the pituitary culture was subjected to disc-PAGE, two distinct bands (GH, R, = 0.25, and PRL, R, = 0.50) were always detected on gels stained with Amido Black 10B through the lo-day period of culture in cu-MEM and Waymouth’s medium. The PRL band was stained consistently the darkest among the protein bands separated. No other bands were detected, except for a very faint band which was observed just in front of the PRL band when gels were stained with Coomassie Blue. These bands were diffused in gels when pituit~ies were cultured in Medium 199. When pituitaries were cultured with i3H]leucine in (u-MEM and the medium was electrophoresed, the major peak of radioactivity was seen at R, = 0.50, where the stained PRL band was present (Fig. 2). The GH band was less radioactive. The distributions of radioimmunoassayable mouse PRL and of receptor-binding ability on gels are shown in Fig. 3. Both these activities are observed only at R, = 0.50, coinciding exactly with the mobility of mouse PRL on the gel. Protein which reacts with the
0
I 2 Distance from origin
( cm I
3
Fig. 3. Distribution of the radioimmunoassayable and receptor-bindable PRL on disc-PAGE gel. 4- or 5-day cultured pituitaries were cultured for 24 h and the medium was electrophoresed. PRL activities in eluates from sliced gels were determined by RIA (e) and RRA (0).
318
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Fig. 4. SDS-PAGE of PRL denatured with (A) and without (B) dithiothreitol. PRL and [3H]PRL were eluted from PRL bands which were separated by disc-PAGE. After electrophoresis in SDS, gels were stained with Coomassie Blue and the radioactivity distribution was determined. Arrows indicate BSA and ovalbumin which were added to eluates as carrier proteins for TCA precipitation. For the culture conditions see Fig. 2.
anti-mouse PRL serum and the mammary PRL receptor site could not be detected in other places on the gel through the lo-day period of culture. The eluted PRL band was denatured in the presence of SDS with or without dithiothreitol and was analysed again by SDS-PAGE. Figs. 4A and 4B show the PRL band stained with Coomassie Blue and the radioactivity distribution of 3H-incorporated protein on the gel. In each figure, stained band and radioactivity were separated as a single band with the same mobility. The molecular weight of mouse PRL was estimated to be 23000 on SDS gels regardless of denaturing in the presence or absence of dithiothreitol. However, PRL and molecular weight marker proteins denatured in the absence of dithiothreitol migrated much faster and diffused more than those denatured in the presence of dithiothreitol.
DISCUSSION Proteins in the medium were separated into two bands, faintly and darkly stained with Arnido Black lOB, and had relative mob&ties of 0.25 and 0.5, respectively, on polyacrylamide gels. Only these two bands were labelled with [3H]leucine. A higher radioactivity was found in the PRL band, and a lower activity in the GH band. Of these two, only the PRL band reacted to anti-mouse PRL serum and also displaced [‘251]ovine
Secreted mouse proiacrin
319
PRL from mouse mammary PRL receptors. The mobility of PRL agrees with those observed in tissue homogenates (Cheever et al., 1969; Lewis et al., 1969). When longer gels were used and stained with Coomassie Blue, one minor band ~grating faster than PRL was found. This charge isomer is probably the deamidated form of mouse PRL (Lewis et al., 1970; Kohmoto, 1975; Farmer et al., 1976). PRL bioactivities, determined by Pigeon crop-sac assays (Cheever et al., 1969; Kohmoto and Bern, 1971; Yanai and Nagasawa, 1974) and by the mouse mammary organ culture (Nicholson, 1969), are principally associated with the PRL band on polyacrylamide gels. Size and charge heterogeneities of PRL have been reported in mice (Sinha and Baxter, 1979a, b) and rats (Dombroske et al., 1976; Aswaroengchai et al., 1978; Wallis et al., 1980). However, the present data show that the PRL receptor-binding activity and immunoreactivity were present at the position of the most darkly stained and heavily 3H-labelled band. Mouse PRL denatured with or without dithiothreitol was separated as a single band with an estimated molecular weight of 23000, as reported by Shore et al. (1978) and Colosi et al. (1982). Mittra (1980) observed intact and cleaved PRL in the culture medium of rat pituitaries on SIX-PAGE. This cleavage of PRL may take place after translation because only a single-chain prePRL was synthesized in a cell-free translation directed by mRNA for rat PRL (Evans et al., 1976). No cleaved form of mouse PRL similar to Mittra’s fraction was observed in our mouse pituitary organ culture system. In analysis of rat and bovine pituitaries or of the medium of bovine pituitary slices cultured in vitro, protein bands equivalent to cleaved PRL could not be found (Zanini and Rosa, 1981). If such cleaved PRL does not bind to either anti-mouse PRL serum or mammary PRL receptors and migrate with a different mobility, these PRLs may be excluded for the subsequent SDS-PAGE in these experiments. However, we observed only two protein bands (GH and PRL) in the stained gel. Cultured pituitaries secrete a large amount of homogeneous PRL into the medium through 10 days. The secreted PRL definitely cross-reacts with anti-mouse PRL serum, binds to mouse mamm~y PRL receptors and migrates with a single mobility both on disc and SDS gels. We conclude, therefore, that the secreted PRL is a single-chain molecule having a molecular weight of 23 000.
ACKNOWLEDGEMENTS We are grateful to NIAMDD
for the supply of ovine PRL (NIH-P-
320
H. Dohi et al.
Sl2), to Mr. T. Kondo for taking care of animals, and to Mrs. K.imiko Tachikawa for typing the manuscript.
REFERENCES Asawaroengchai, H., Russel, SM. and Nicoll, C.S. (1978) Endocrinology 102, 407-414. Cheever, E.V. and Lewis, U.J. (1969) Endocrinology 85,465-473. Cheever, E.V., Seavey, B.K. and Lewis, U.J. (1969) Endocrinology 85, 698-703. Colosi, P., Marr, G., Lopez, J., Ogren, L. and Talamantes, F. (1982) Proc. Nat]. Acad. Sci. (U.S.A.) 79, 771-775. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. Dombroske, O.C., Payne, P. and Fran@ W.L. (1976) Fed. Proc. 35, 219 (abstract). Evans, G.A. and Rosenfeld, M.G. (1976) 3. Biol. Chem. 215, 2842-2847. Evans, G.A., Hucko, J. and Rosenfeld, M.G. (1977) Endocrinology 101, 1807-1814. Farmer, S.W., Bewley, T.A., Russel, SM. and Nicoll, C.S. (1976) Biochim. Biophys. Acta 437, 562-570. Harigaya, T., Sakai, S., Kohmoto, K. and Shoda, Y. (1982) J. Endocrinol. 94, 149-155. Kohmoto, K. (1975) Endocrinol. Jpn. 22, 465-469. Kohmoto, K. and Bern, H.A. (1971) Proc. Sot. Exp. Biol. Med. 137, 807-809. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Lewis, U.J., Littreia, M. and Cheever, E.V. (1969) Endocrinology 85, 690-697. Lewis, U.J., Cheever, E.V. and Hopkins, W.C. (1970) Biochim. Biophys. Acta 214,498-508. Maure, R.A., Stone, R. and Gorski, J. (1976) J. Biol. Chem. 251, 2801-2807. Mittra, I. (1980) B&hem. Biophys. Res. Commun. 95, 1750-1759. Nicholson, P.M. (1969) J. Endocrinol. 44, 459-460. Omstein, 0. (1964) Ann. N.Y. Acad. Sci. 121, 321-349. Parlow, A.F. and Shome, B. (1976) Fed. Proc. 35, 219. Sakai, S., Enami, J., Nandi, S. and Banerjee, M.R. (1978) Mol. Cell. Endocrinol. 12, 285-298. Shore, L.F., Shine, N.R. and Talamantes, F. (1978) Biochim. Biophys. Acta 537, 336-347. Sinha, Y.N. (1980) Endocrinology 107, 1959-1969. Sinha, Y.N. and Baxter, S.R. (1979a) Biochem. Biophys. Res. Commun. 86, 325-330. Sinha, Y.N. and Baxter, S.R. (1979b) J. Endocrinol. 81, 229-314. Wallis, M,, Daniels, M. and Ellis, S.A. (1980) Biochem. J. 189, 605-614. Yanai, R. and Nagasawa, H. (1974) J. Endocrinol. 62, 685-686. Zanini, A. and Rosa, P. (1981) Mol. Cell. EndocrinoI. 24, 165-179. Zanini, A., Giannattasio, G. and Meldolesi, J. (1974) Endocrinology 94, 594-598.