Molec. Biol. Rep. Vol. 5, 3: 175-179, 1979
THE SYNTHESIS AND SECRETION OF HUMAN CHORIONIC GONADOTROPIN BY TISSUE SLICES FROM FIRST TRIMESTER PLACENTAS 1
R. FOLMAN 1 , J. ILAN , J. SHIKLOSH3 , N. DE GROOT 1 , S. SEGAL4 & A.A. HOCHBERG 1
1Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 2Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio, USA 3Department of Obstetrics and Gynaecology, Bikur Cholim Hospital, Jerusalem, Israel 4Population Council, Rockefeller Foundation, New York, USA (Received February 12, 1979)
Abstract
Placental tissue slices' from first trimester placentas synthesize and secrete proteins labeled by radioactive glucosamine are preferentially secreted as compared to proteins in general. One of the proteins synthesized and secreted is hCG. Processing and secretion of proteins, including hCG, by the tissue slices need a two-hour period. Both secretion and glycosylation of the protein can take place independently of protein synthesis. A method was developed for the specific determination of newly synthesized radioactive hCG in placental tissue.
Introduction
The placenta is a rapidly develoving tissue, which besides its own proteins synthesizes considerable quantities of polypeptide hormones which are secreted into the maternal blood stream. The relative amounts of the major placental polypeptide hormones hCG (human chorionic gonadotropin) and hPL (human placental lactogen) changes drastically during pregnancy (1) and the highest level of hCG is found in the first trimester of pregnancy. The placental hormones, including hCG, play an important role in the maintenance of pregnancy and the well-being of the foetus (2-4). It is of great importance to relate the protein synthesizing capacity of the placenta to certain deviations in the normal course of foetal development. With this purpose in mind, we are studying protein synthesis and secretion by placental tissue slices obtained from human placentas at different stages of pregnancy. Other investigators have
shown that placental tissue slices are active in protein synthesis in general and are able to synthesize hCG (5-8).
Materials and methods
L[4,5-aH] leucine (specific radioactivity 54 Ci/ mmole), L[ s s S] methionine (460 Ci/mmole), L[2, 3, 4, 5-all] proline (105 Ci/mmole), D-[6-aH] glucos.amine HCI (19 Ci/mmole) and [12s I] sodium iodide (carrier-free) were purchased from the Radiochemical Centre, Amersham, England. Antibodies to hCG, hCG-a and hCG-~ prepared from rabbit serum, as well as purified hCG, hCG-a and hCG-15 were the kind gift of Dr. S.S. Koide The Population Council, Rockefeller University, New york. hCG, hCG-a and hCG-15were chemically iodinated with chloramine T according to Letchworth (9). The amino acid incorporation activity of tissue slices was determined as follows: First-trimester placental tissue was obtained from therapeutic abortion, transported in 0.9% NaC1 in the cold, processed within 30 min. and incubated in KrebsRinger bi-carbonate buffer (KRB) containing 0.5 mg/ ml glucose at 37~ for 10 min. The minced tissue Was collected by filtration and 250 mg tissue portions were incubated in 2.5 ml of KRB buffer pH 7.4, containing 100 units/ml sodium penicillin G, 5 /.tg/ mI streptomycin sulfate, a mixture of 20 non-radioactive amino acids, 0.1 mM each; 10/~ ci of one-of the following compounds: [all] leucine, [all] proline, [3 s S] methionine and [3 H] glucosamine - added at zero time. The incubation was carried out in a 175
shaker bath at 120 cycles/min, 37~ in an atmosphere of 95% O2 and 5% CO2. After incubation, the radioactive substrate was diluted by the addition of 10 /amoles of the appropriate non-radioactive compound, the tissue slices were separated from the medium by centrifugation at 25000 x g for 10 min. The tissue slices were homogenized in 0.25 M sucrose containing Tris buffer (Tris-HC1, pH 7.4, 50 mM; KCI 25 mM; MgCI2 10 mM; NH4C1 100 mM; EDTA 0.5 mM). In some of the experiments subcellular fractions were isolated from the homogenate according to Gal et al. (10). The radioactivity of the hot 5% TCA insoluble material of the homogenate and subcellular fractions was determined according to Bollum et al. (11). Protein was determined according to Lowry et al. (12). The hCG content of the tissue slices and the medium was determined as follows: 500/al of assay buffer at pH 7.8 containing sodium phosphate, 0.01 M; NaCI 0.15 M; sodium azide 0.1%; BSA 1%; 100 ttl of EDTA 0.1 M, pH 7.0; 20/.d of sample of hCG standard (0-50 IU); [12s i] hCG, 30,000 cpm and 200/al of rabbit anti-hCG serum diluted in assay buffer containing 1% normal rabbit serum. The assay mixture was incubated at 4~ overnight, anti-rabbit7-globulin was added and the mixture incubated for an additional 24 hrs at 4~ The mixture was centrifuged and the radioactivity of the precipitate counted in a Packard 7 spectrometer. SDS gel dectrophoresis was done according to Laemmli (13). Lactic dehydrogenase was determined according to Kornberg (14).
Results and discussion Tissue slices from first trimester placentas incorporate amino acids into proteins and excrete a part of these proteins into the medium (Fig. 1). When labeled methionine or proline were used instead of radioactive leucine similar results were obtained. The incubation mixture contained 20 amino acids at 0.1 mM (including the labeled amino acids) and under these conditions the incorporation was nearly linear for at least eight hours. However, hot TCA insolubel radioactive material could be detected in the medium only after a 2-hr incubation. This is very likely, due to the time necessary for the transport, processing and secretion of the protein after the synthesis of its peptide backbone. From the results it can be calculated that after an eight-hour incubation period, approximately 5% of the newly 176
ac
/
2
/
/
4
| 6
i 8 ht
Fig. 1. Protein synthesis by and protein secretion from human placental tissue slices. 9
o, tissue
slices
o o, medium [3H] leueine was added as the radioactive substrate. synthesized labeled protein appears in the cell-free medium. The appearance of hot TCA insoluble radioactive material in the medium is not due to tissue damage or leakage of proteins from the tissue cells. There was no measurable increase of protein in the medium during the incubation. Moreover, during the incubation period no lactic dehydrogenase activity was released into the medium. Tissue slices have a considerable lactic dehydrogenase activity and even the release of 1% of the tissue lactic dehydrogenase activity into the medium should be easily detectable. Tissue slices were incubated with radioactive amino acids and the homogenate was fraetionated and the specific activity of the proteins of the subcellular fractions was determined. As can be seen from Fig. 2A, the proteins of the rough membrane fractions had, after a short incubation period (1 min), a specific activity of at least three times that of the proteins of the smooth membrane or the free polyribosomal fraction, but on extending the incubation, the specific activity of the smooth membrane proteins was equal to those of the rough membrane (30 min) and was one and a half times as high after 6 hrs (Fig. 2B). The proteins of different fractions, after a labeling experiment with [as S] methionine, were run on an SDS gel electrophoresis and autoradiograms prepared. Radioactive proteins with an electrophoretic mobility identical to that of ~ and 13 hCG could be detected in the rough and smooth membrane fractions and in the incubation medium. Fig. 3 shows the amount of hCG, as determined by radioimmunoassay, in the tissue slices and in the incubation medium. The hCG content of the tissue remains more or less constant b u t that of
RM SM
!
FP
I 34
1
I SO
2
3
4
rain
5
6
hr
Figs. 2A and 2B. The specific activity of labeled protein in subcellular fractions isolated from tissue slices labeled in vitro.
The radioactive amino acid used was [3 H] leueine.
MEI~UM 6O
S
1
2
3
4
Fig. 3. hCG content in tissue slices and incubation medium as measured by radioimmunoassay.
the medium increases steadily, and after an incubation period of 5 hrs, the hCG content of the medium is equal to that of the tissue. Therefore, there is a 100% increase in the amount of hCG in the incubation mixture and thus it is clear that d e n o v o synthesis of hCG occurs during the incubation period. It should be noted, however, that hCG appears in the medium without a long period notwithstanding that radioactive hot TCA insoluble material made its ftrst appearance in the medium only after a 2 hrs lag period, hCG is therefore continuously synthesized and secreted during the incubation period. In order to determine exactly the amount of newly synthesized hCG present in the tissue slices at any time of the incubation period, we added, after a labeling experiment, excess carrier hCG to the homogenate. Antibodies were added in amounts such that optimal precipitation of hCG was obtained. After incubation at 4~ for 48 hrs, anti-rabbit 7-globulin was added followed by an additional 48 hrs of incubation. The mixture was centrifuged, the precipitate taken up in 2% SDS, and an auto-radiogram was prepared for the gel electrophoretogram. The autoradiogram showed the presence of several proteins besides hCG-a and hCG-/L It was therefore decided to enrich hCG in the tissue homogenate and in the medium before antibody precipitation. The procedure for hCG enrichment was developed using [12 s I] labeled hCG as a tracer. Triton X-100 (final concentration 1%) and [~2sI] hCG were added to the 9,000 xg supernatant, prepared from tissue slices homogenates, after an incubation without a radioactive amino acid. The mixtures were centrifuged at. 100,000 xg for 30 min. Two volumes of ethanol were added to the supernatants which were centrifuged for 10 min at 30,000 xg. The sediments were suspended in HzO (1:10 v/v), homogenized and centrifuged again. More than 85% of the radioactive material was found in the aqueous medium. The supernatants were chromatographed on Bio-gel P-60 columns (1.0 x I0 cm) which were equilibrated and eluted at room temperature with 0.01 M sodium phosphate, pH 7.8, containing 0.15 M NaC1. One ml fractions were collected, lyophilized, and dissolved in water, hCG was detected in fractions 3 and 4. Tissue slices were labeled with [s s S] methionine. Both the medium and the tissue slices homogenate were processed as described above (without [12sI] hCG added) and hCG in fractions 3 and 4 was precipitated using the double antibody precipitation method. The precipitate was dissolved in 2% SDS 177
solution, electrophoresed on an SDS-polyacrylamide gel, and the autoradiogram showed almost exclusively the presence of only two protein bands with electrophoretic mobilities identical to that of hCG-a and HCG-~. From the results it could be calculated that after a six hour incubation period, 5% of the labeled protein in the tissue slices and 18% in the medium is hCG. Radioactive material could be detected in fractions 3 and 4 from the medium only after a 2 hour incubation period, proving that no radioactive hCG is secreted into the medium during the first two hours of incubation: This finding is in complete agreement with the results shown in Fig. 1. In order to determine the correlation between synthesis of the protein backbone and the processing and secretion from the cell, we added protein synthesis inhibitors to the incubation mixture. Immediately after the addition of puromycin or cycloheximide, protein synthesis comes to a complete standstill. We even found a drop in the hot TCA insoluble radioactive "material in the tissue homogenates (Fig. 4A). The secretion of hot TCA insoluble radioactive material into the medium continues for at least one hour after the addition of the inhibitors, but afterwards drops to zero (Fig. 4B). If the medium containing the inhibitor is replaced by a fresh medium, protein synthesis immediately fully recovers but the secretion of hot TCA insoluble material into the medium will start again only after a lag time of 1-2 hrs (results not shown). Our conclusion is that secretion can take place in the complete absence of protein synthesis. As hCG is a glycoprotein, tissue slices were incubated with [3HI glucosamine. Fig. 5 shows that ~glucosamine is incorporated by the tissue slices into hot TCA insoluble material and secreted after a 2-hr lag period. It can be calculated that after 6 hrs of incubation, 30% of the hot TCA insoluble material appeared in the medium when glucosamine was the radioactive precursor, as compared to 5% when the tissue was incubated with radioactive leucine under the same conditions. From these results one can conclude that glycoproteins are preferentially secreted from the tissue slices and that the lag period in the appearance of radioactive material in the medium is due to a step in the processing or secretion of the glycoprotein after the incorporation of the labeled glucosamine-derived sugar residues into the glycoprotein (N-acetyl glucosamine and sialic acid). When puromycin was added to the incubation medium, incorporation of labeled glucosamine was inhibited (results not shown). Nevertheless, although 178
A tissue
.//.../.CONTROL
E
~ /~"/
i
----a
~'~~ ~ ~__
CHI (4
---
- ...... 2
PURO ~4 hr)
~
4
I 8
hr)
PURO ;CHI ( 0~ hr
CHI-4 l~r
PURO-O
2
4
6
8
h,
Figs. 4A alld 4B. TI/e effect of protein synthesis irthibitors on the synthesis (A) and on the secretion (B) of proteins by the tissue slices. Inhibitors were added at zero time or after 4 hrs of incubation. [3H] leucine was used as the radioactive substrate.
tissue
| E
medium
2
4
5 hr
Fig. 5. The rate of synthesis and secretion of glycoprotein by the tissue slices. [3H] glucosamine was used as substrate.
protein synthesis was immediately partly inhibited, glyeosylation continued at a lower rate for 1 hour, indicating that polypeptides c o n t i n u e d to be glyeosylated, even in the absence o f de novo polypeptide synthesis.
Acknowledgements This work was supported b y grant No. CB 78.22x/ ICCR from the Population Council, the Rockefeller University, New York, USA. We thank Mrs. Tamar Schneider for her excellent technical assistance.
References 1. Brody, S. and Carlstr/Sm, C. J. Clin. Endocrinol. Metab. 22, 546-574 (1962). 2. Neil, J.D., Johansson, E.D.B. and Knobil, E. Endocrinology 84, 45-48 (1969). 3. Neil, J.D. and Knobil, E. Endocrinology 90, 34-38 (1971). 4. Strott, C.A. Yoshimi, T., Ross, G.T. and Lispell, M.B. J. Clin. Endocrinol. 29, 1157-1167 (1969). 5. Gitlin, D. and Biasucci, A. Clin. Endocrinol. Metab. 29, 926-935 (1969). 6. Benagiano, G., Pala, A., Meirinho, M. and Ermini, M. J. Endocrinol. 55, 387-396 (1972). 7. Patrito, L.C., Flury, A., Rosato, J. and Martin, A. Hoppe Seyler's Z. Physiol. Chem. 354, 1129-1132 (1973). 8. Maruo, T., Ashitaka, Y., Mochizuki, M. and Tojo, S. Endocrinol. Jap. 21,499-505 (1974). 9. Letchworth, A.T., Boardman, R., Bristow, C., London, J. and Chard, T. J. of Obst. Gynaecol. 78, 535-541 (1971). 10. Gal, A.L., Folman, R., Czosnek, H.H., Shiklosh, J., de Groot, N. and Hoehberg, A.A., Life Sci. 21, 779788(1977). 11. BoUum, F.J., in Procedures in Nucleic Acid Research (Eds. G.L. Cantoni and D.R. Davies), Harper and Row, New York, pp. 296-302 (1965). t 2. Lowry, O.H., Rosebrough, N 3 , Farr, A.L. and Randall, R.J., J. Biol. Chem. 193,265-275 (1951). 13. Laemmli, U.K., Nature 227,680-685 (1970). 14. Kornberg, A., Methods in Enzymol. 1,441-443 (1955).
179
THE SYNTHESIS AND SECRETION OF HUMAN CHORIONIC GONADOTROPIN BY TISSUE SLICES FROM FIRST TRIMESTER PLACENTAS 1
R. FOLMAN 1 , J. ILAN , J. SHIKLOSH3 , N. DE GROOT 1 , S. SEGAL4 & A.A. HOCHBERG 1
1Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 2Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio, USA 3Department of Obstetrics and Gynaecology, Bikur Cholim Hospital, Jerusalem, Israel 4Population Council, Rockefeller Foundation, New York, USA (Received February 12, 1979)
Abstract
Placental tissue slices' from first trimester placentas synthesize and secrete proteins labeled by radioactive glucosamine are preferentially secreted as compared to proteins in general. One of the proteins synthesized and secreted is hCG. Processing and secretion of proteins, including hCG, by the tissue slices need a two-hour period. Both secretion and glycosylation of the protein can take place independently of protein synthesis. A method was developed for the specific determination of newly synthesized radioactive hCG in placental tissue.
Introduction
The placenta is a rapidly develoving tissue, which besides its own proteins synthesizes considerable quantities of polypeptide hormones which are secreted into the maternal blood stream. The relative amounts of the major placental polypeptide hormones hCG (human chorionic gonadotropin) and hPL (human placental lactogen) changes drastically during pregnancy (1) and the highest level of hCG is found in the first trimester of pregnancy. The placental hormones, including hCG, play an important role in the maintenance of pregnancy and the well-being of the foetus (2-4). It is of great importance to relate the protein synthesizing capacity of the placenta to certain deviations in the normal course of foetal development. With this purpose in mind, we are studying protein synthesis and secretion by placental tissue slices obtained from human placentas at different stages of pregnancy. Other investigators have
shown that placental tissue slices are active in protein synthesis in general and are able to synthesize hCG (5-8).
Materials and methods
L[4,5-aH] leucine (specific radioactivity 54 Ci/ mmole), L[ s s S] methionine (460 Ci/mmole), L[2, 3, 4, 5-all] proline (105 Ci/mmole), D-[6-aH] glucos.amine HCI (19 Ci/mmole) and [12s I] sodium iodide (carrier-free) were purchased from the Radiochemical Centre, Amersham, England. Antibodies to hCG, hCG-a and hCG-~ prepared from rabbit serum, as well as purified hCG, hCG-a and hCG-15 were the kind gift of Dr. S.S. Koide The Population Council, Rockefeller University, New york. hCG, hCG-a and hCG-15were chemically iodinated with chloramine T according to Letchworth (9). The amino acid incorporation activity of tissue slices was determined as follows: First-trimester placental tissue was obtained from therapeutic abortion, transported in 0.9% NaC1 in the cold, processed within 30 min. and incubated in KrebsRinger bi-carbonate buffer (KRB) containing 0.5 mg/ ml glucose at 37~ for 10 min. The minced tissue Was collected by filtration and 250 mg tissue portions were incubated in 2.5 ml of KRB buffer pH 7.4, containing 100 units/ml sodium penicillin G, 5 /.tg/ mI streptomycin sulfate, a mixture of 20 non-radioactive amino acids, 0.1 mM each; 10/~ ci of one-of the following compounds: [all] leucine, [all] proline, [3 s S] methionine and [3 H] glucosamine - added at zero time. The incubation was carried out in a 175
shaker bath at 120 cycles/min, 37~ in an atmosphere of 95% O2 and 5% CO2. After incubation, the radioactive substrate was diluted by the addition of 10 /amoles of the appropriate non-radioactive compound, the tissue slices were separated from the medium by centrifugation at 25000 x g for 10 min. The tissue slices were homogenized in 0.25 M sucrose containing Tris buffer (Tris-HC1, pH 7.4, 50 mM; KCI 25 mM; MgCI2 10 mM; NH4C1 100 mM; EDTA 0.5 mM). In some of the experiments subcellular fractions were isolated from the homogenate according to Gal et al. (10). The radioactivity of the hot 5% TCA insoluble material of the homogenate and subcellular fractions was determined according to Bollum et al. (11). Protein was determined according to Lowry et al. (12). The hCG content of the tissue slices and the medium was determined as follows: 500/al of assay buffer at pH 7.8 containing sodium phosphate, 0.01 M; NaCI 0.15 M; sodium azide 0.1%; BSA 1%; 100 ttl of EDTA 0.1 M, pH 7.0; 20/.d of sample of hCG standard (0-50 IU); [12s i] hCG, 30,000 cpm and 200/al of rabbit anti-hCG serum diluted in assay buffer containing 1% normal rabbit serum. The assay mixture was incubated at 4~ overnight, anti-rabbit7-globulin was added and the mixture incubated for an additional 24 hrs at 4~ The mixture was centrifuged and the radioactivity of the precipitate counted in a Packard 7 spectrometer. SDS gel dectrophoresis was done according to Laemmli (13). Lactic dehydrogenase was determined according to Kornberg (14).
Results and discussion Tissue slices from first trimester placentas incorporate amino acids into proteins and excrete a part of these proteins into the medium (Fig. 1). When labeled methionine or proline were used instead of radioactive leucine similar results were obtained. The incubation mixture contained 20 amino acids at 0.1 mM (including the labeled amino acids) and under these conditions the incorporation was nearly linear for at least eight hours. However, hot TCA insolubel radioactive material could be detected in the medium only after a 2-hr incubation. This is very likely, due to the time necessary for the transport, processing and secretion of the protein after the synthesis of its peptide backbone. From the results it can be calculated that after an eight-hour incubation period, approximately 5% of the newly 176
ac
/
2
/
/
4
| 6
i 8 ht
Fig. 1. Protein synthesis by and protein secretion from human placental tissue slices. 9
o, tissue
slices
o o, medium [3H] leueine was added as the radioactive substrate. synthesized labeled protein appears in the cell-free medium. The appearance of hot TCA insoluble radioactive material in the medium is not due to tissue damage or leakage of proteins from the tissue cells. There was no measurable increase of protein in the medium during the incubation. Moreover, during the incubation period no lactic dehydrogenase activity was released into the medium. Tissue slices have a considerable lactic dehydrogenase activity and even the release of 1% of the tissue lactic dehydrogenase activity into the medium should be easily detectable. Tissue slices were incubated with radioactive amino acids and the homogenate was fraetionated and the specific activity of the proteins of the subcellular fractions was determined. As can be seen from Fig. 2A, the proteins of the rough membrane fractions had, after a short incubation period (1 min), a specific activity of at least three times that of the proteins of the smooth membrane or the free polyribosomal fraction, but on extending the incubation, the specific activity of the smooth membrane proteins was equal to those of the rough membrane (30 min) and was one and a half times as high after 6 hrs (Fig. 2B). The proteins of different fractions, after a labeling experiment with [as S] methionine, were run on an SDS gel electrophoresis and autoradiograms prepared. Radioactive proteins with an electrophoretic mobility identical to that of ~ and 13 hCG could be detected in the rough and smooth membrane fractions and in the incubation medium. Fig. 3 shows the amount of hCG, as determined by radioimmunoassay, in the tissue slices and in the incubation medium. The hCG content of the tissue remains more or less constant b u t that of
RM SM
!
FP
I 34
1
I SO
2
3
4
rain
5
6
hr
Figs. 2A and 2B. The specific activity of labeled protein in subcellular fractions isolated from tissue slices labeled in vitro.
The radioactive amino acid used was [3 H] leueine.
MEI~UM 6O
S
1
2
3
4
Fig. 3. hCG content in tissue slices and incubation medium as measured by radioimmunoassay.
the medium increases steadily, and after an incubation period of 5 hrs, the hCG content of the medium is equal to that of the tissue. Therefore, there is a 100% increase in the amount of hCG in the incubation mixture and thus it is clear that d e n o v o synthesis of hCG occurs during the incubation period. It should be noted, however, that hCG appears in the medium without a long period notwithstanding that radioactive hot TCA insoluble material made its ftrst appearance in the medium only after a 2 hrs lag period, hCG is therefore continuously synthesized and secreted during the incubation period. In order to determine exactly the amount of newly synthesized hCG present in the tissue slices at any time of the incubation period, we added, after a labeling experiment, excess carrier hCG to the homogenate. Antibodies were added in amounts such that optimal precipitation of hCG was obtained. After incubation at 4~ for 48 hrs, anti-rabbit 7-globulin was added followed by an additional 48 hrs of incubation. The mixture was centrifuged, the precipitate taken up in 2% SDS, and an auto-radiogram was prepared for the gel electrophoretogram. The autoradiogram showed the presence of several proteins besides hCG-a and hCG-/L It was therefore decided to enrich hCG in the tissue homogenate and in the medium before antibody precipitation. The procedure for hCG enrichment was developed using [12 s I] labeled hCG as a tracer. Triton X-100 (final concentration 1%) and [~2sI] hCG were added to the 9,000 xg supernatant, prepared from tissue slices homogenates, after an incubation without a radioactive amino acid. The mixtures were centrifuged at. 100,000 xg for 30 min. Two volumes of ethanol were added to the supernatants which were centrifuged for 10 min at 30,000 xg. The sediments were suspended in HzO (1:10 v/v), homogenized and centrifuged again. More than 85% of the radioactive material was found in the aqueous medium. The supernatants were chromatographed on Bio-gel P-60 columns (1.0 x I0 cm) which were equilibrated and eluted at room temperature with 0.01 M sodium phosphate, pH 7.8, containing 0.15 M NaC1. One ml fractions were collected, lyophilized, and dissolved in water, hCG was detected in fractions 3 and 4. Tissue slices were labeled with [s s S] methionine. Both the medium and the tissue slices homogenate were processed as described above (without [12sI] hCG added) and hCG in fractions 3 and 4 was precipitated using the double antibody precipitation method. The precipitate was dissolved in 2% SDS 177
solution, electrophoresed on an SDS-polyacrylamide gel, and the autoradiogram showed almost exclusively the presence of only two protein bands with electrophoretic mobilities identical to that of hCG-a and HCG-~. From the results it could be calculated that after a six hour incubation period, 5% of the labeled protein in the tissue slices and 18% in the medium is hCG. Radioactive material could be detected in fractions 3 and 4 from the medium only after a 2 hour incubation period, proving that no radioactive hCG is secreted into the medium during the first two hours of incubation: This finding is in complete agreement with the results shown in Fig. 1. In order to determine the correlation between synthesis of the protein backbone and the processing and secretion from the cell, we added protein synthesis inhibitors to the incubation mixture. Immediately after the addition of puromycin or cycloheximide, protein synthesis comes to a complete standstill. We even found a drop in the hot TCA insoluble radioactive "material in the tissue homogenates (Fig. 4A). The secretion of hot TCA insoluble radioactive material into the medium continues for at least one hour after the addition of the inhibitors, but afterwards drops to zero (Fig. 4B). If the medium containing the inhibitor is replaced by a fresh medium, protein synthesis immediately fully recovers but the secretion of hot TCA insoluble material into the medium will start again only after a lag time of 1-2 hrs (results not shown). Our conclusion is that secretion can take place in the complete absence of protein synthesis. As hCG is a glycoprotein, tissue slices were incubated with [3HI glucosamine. Fig. 5 shows that ~glucosamine is incorporated by the tissue slices into hot TCA insoluble material and secreted after a 2-hr lag period. It can be calculated that after 6 hrs of incubation, 30% of the hot TCA insoluble material appeared in the medium when glucosamine was the radioactive precursor, as compared to 5% when the tissue was incubated with radioactive leucine under the same conditions. From these results one can conclude that glycoproteins are preferentially secreted from the tissue slices and that the lag period in the appearance of radioactive material in the medium is due to a step in the processing or secretion of the glycoprotein after the incorporation of the labeled glucosamine-derived sugar residues into the glycoprotein (N-acetyl glucosamine and sialic acid). When puromycin was added to the incubation medium, incorporation of labeled glucosamine was inhibited (results not shown). Nevertheless, although 178
A tissue
.//.../.CONTROL
E
~ /~"/
i
----a
~'~~ ~ ~__
CHI (4
---
- ...... 2
PURO ~4 hr)
~
4
I 8
hr)
PURO ;CHI ( 0~ hr
CHI-4 l~r
PURO-O
2
4
6
8
h,
Figs. 4A alld 4B. TI/e effect of protein synthesis irthibitors on the synthesis (A) and on the secretion (B) of proteins by the tissue slices. Inhibitors were added at zero time or after 4 hrs of incubation. [3H] leucine was used as the radioactive substrate.
tissue
| E
medium
2
4
5 hr
Fig. 5. The rate of synthesis and secretion of glycoprotein by the tissue slices. [3H] glucosamine was used as substrate.
protein synthesis was immediately partly inhibited, glyeosylation continued at a lower rate for 1 hour, indicating that polypeptides c o n t i n u e d to be glyeosylated, even in the absence o f de novo polypeptide synthesis.
Acknowledgements This work was supported b y grant No. CB 78.22x/ ICCR from the Population Council, the Rockefeller University, New York, USA. We thank Mrs. Tamar Schneider for her excellent technical assistance.
References 1. Brody, S. and Carlstr/Sm, C. J. Clin. Endocrinol. Metab. 22, 546-574 (1962). 2. Neil, J.D., Johansson, E.D.B. and Knobil, E. Endocrinology 84, 45-48 (1969). 3. Neil, J.D. and Knobil, E. Endocrinology 90, 34-38 (1971). 4. Strott, C.A. Yoshimi, T., Ross, G.T. and Lispell, M.B. J. Clin. Endocrinol. 29, 1157-1167 (1969). 5. Gitlin, D. and Biasucci, A. Clin. Endocrinol. Metab. 29, 926-935 (1969). 6. Benagiano, G., Pala, A., Meirinho, M. and Ermini, M. J. Endocrinol. 55, 387-396 (1972). 7. Patrito, L.C., Flury, A., Rosato, J. and Martin, A. Hoppe Seyler's Z. Physiol. Chem. 354, 1129-1132 (1973). 8. Maruo, T., Ashitaka, Y., Mochizuki, M. and Tojo, S. Endocrinol. Jap. 21,499-505 (1974). 9. Letchworth, A.T., Boardman, R., Bristow, C., London, J. and Chard, T. J. of Obst. Gynaecol. 78, 535-541 (1971). 10. Gal, A.L., Folman, R., Czosnek, H.H., Shiklosh, J., de Groot, N. and Hoehberg, A.A., Life Sci. 21, 779788(1977). 11. BoUum, F.J., in Procedures in Nucleic Acid Research (Eds. G.L. Cantoni and D.R. Davies), Harper and Row, New York, pp. 296-302 (1965). t 2. Lowry, O.H., Rosebrough, N 3 , Farr, A.L. and Randall, R.J., J. Biol. Chem. 193,265-275 (1951). 13. Laemmli, U.K., Nature 227,680-685 (1970). 14. Kornberg, A., Methods in Enzymol. 1,441-443 (1955).
179
