Biochimica et Biophysiea Acta, 493 (1977) 332-339 © Elsevier/North-Holland Biomedical Press BBA 37715
M I C R O H E T E R O G E N E I T Y OF H U M A N PLACENTAL LACTOGEN SHOWING D I F F E R E N T PATTERNS IN INDIVIDUALS
MEERA CHATTERJEE, EMIL M. LAGA, CHARLES C. MERRILL and H A M I S H N. M U N R O
Physiological Chemistry Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of" Technology, Cambridge, Mass. 02139 (U.S.A.) (Received December 21st, 1976)
SUMMARY
Samples of human placental lactogen, obtained either as a standard pooled preparation or prepared from individual placentas, were shown to migrate as a single band on acrylamide gel electrophoresis. When subjected to isoelectric focusing in polyacrylamide gels, the pooled sample was resolved into bands at pI values 5.0, 5.5, 5.8, 6.0, 6.1 and 6.2. Different batches of the standard pooled sample gave different proportions of each isoprotein species. Isolation and refocusing of individual bands did not alter the pI of each. Treatment with urea or with p-chloromercuribenzoate did not eliminate microheterogeneity seen on isofocusing, indicating that the observed heterogeneity is probably not due to conformational differences or to restriction of molecular shape by disulfide bonds. It was shown by immunodiffusion that all the isofocusing bands reacted similarly against a common antibody to human placental lactogen. When placental lactogen was extracted from individual full term human placentas, the same isoprotein bands were observed but their proportions varied markedly from one placenta to another, and not all bands were present. Thus human placental lactogen displays considerable microheterogeneity which varies with individual placentas.
INTRODUCTION
Heterogeneity among placental proteins has been described for alkaline phosphatase [1], malate and lactate dehydrogenases [2], leucine aminopeptidase [3-5], as well as the other major peptide hormone, human choriogonadotropin [6, 7]. Variation in the latter was thought to be due to differences in its sialic acid content, but differences in the amino acid sequence at the N-terminal end of the a-subunit have since been observed [8]. Belleville et al. [9-12] have characterised human placental lactogen by polyacrylamide gel electrophoresis, describing "slow" and "fast" bands. These were also seen on I polyacrylamide isofocusing and were ascribed to the degree of oxidation of disulfide interchain linkages. Thus only one primary structure was considered to be present. However, Chrambach and his colleagues have shown that human pituitary prolactin [13] and human growth hormone [14, 15], which display structural homology with human placental lactogen [16], show multiple isoproteins by iso-
333 electric focusing. This suggests that isofocusing of human placental lactogen may also reveal microheterogeneity. We have accordingly used isoelectric focusing to examine batch commercial preparations of pooled human placental lactogen and also human placental lactogen prepared from individual placentas. This demonstrated marked microheterogeneity which varied in pattern from one placenta to another, and which cannot be attributed to disulfide linkages. MATERIALS AND METHODS
Preparation of human placental lactogen. Human placental lactogen which had been extracted from pooled term placentas by the method of Friesen [17] and stated to be 95 ~ pure and homogeneous by polyacrylamide electrophoresis, was purchased from Nutritional Biochemicals, (Batch Numbers 3359 and 5501) and from Lederle ("Purified Placental Protein"). In addition, human placental lactogen was extracted by us from individual placentas obtained at normal full term deliveries. The extraction and purification of the peptide hormone followed Kumai's [18] modification of the original Friesen procedure, with substitution of DEAE-cellulose for ECTEOLAcellulose. Each fresh term placenta was homogenized and then extracted at pH 8.5 followed by adjustment of the supernatant fraction to pH 4.6. The supernatant after this treatment was brought to 50~o saturation with (NH4)2SO4 and the precipitate was harvested. Following dialysis to remove the ammonium sulfate, the solution was applied to a Sephadex G-100 column equilibrated with 0.1 M (NH4)2CO3. The retarded fraction was then applied to a DEAE-cellulose column and eluted with increasing concentrations of NH4HCO3 (0.05, 0.1 and 0.3 M). The 0.3 M fraction contained human placental lactogen, and was lyophilized. This procedure was monitored by radioimmunoassay with the Schwarz-Mann assay kit to identify the most active fractions for human placental lactogen activity. Later, the Ouchterlony immunodiffusion test using anti-human placental lactogen sera prepared in the laboratory was also employed. Anti-human placental lactogen serum. New Zealand White rabbits (2.5 kg) were immunized with the commercial human placental lactogen sample, by injecting 1 mg human placental lactogen mixed with 1 ml complete Freund's adjuvant weekly for 5 weeks. The serum harvested at the end of 5 weeks was found to have a high titer against human placental lactogen. This potent antiserum was used to check the stages in human placental lactogen purification from individual placentas by the Ouchterlony double immunodiffusion method [19] and for identifying the individual bands obtained on isofocusing. Electrophoresis of human placental lactogen. All preparations were subjected to electrophoresis in 5 ~o polyacrylamide gels according to the method of Ornstein [20] and Davis [21]. A continuous Tris/glycine buffer system (pH 8.3) was used and electrophoresis was carried out at 2 mA/gel for 7-8 h or 5 mA/gel for 5-6 h at 25 °C or at 2 mA/gel for 17 h at 4 °C. The gels were scanned by a Gilford recording spectrophotometer at 280 nm. Isoelectric focusing of human placental lactogen. The apparatus used (Metalloglass, Inc. Boston, Mass.) was identical to that of Righetti and Drysdale [22]. Polyacrylamide gels (4~, with 0.8 ~ bis) containing 1.5 ~ ampholyte (LKB Produkter, AB), pH 3-10, 4-6, or 5-7, were used. Sample loads ranged from 5 to 100 #g protein
334 per gel (3 X 100 mm). It was determined that separation of 20 #g human placental lactogen was optimum for protein staining and 5/~g for immunological detection. An initial current of 0.5 mA/gel was applied and voltage maintained at 200 V for 17 h. A circulating coolant, 5 ~o ethylene glycol, was used to maintain the temperature of the gels at 0-4 °C. The anolyte used was 0.01 M HaPO4 and the catholyte 0.02 M NaOH. After brief electrolysis (1 mA/tube for 10 min) to discharge persulfate, the samples were applied through the catholyte onto the top of the gel in a dense buffered solution ( 5 ~ ampholyte) titrated to the pH at which the proteins were in anionic form. Horse spleen ferritin (pl 4.2-4.6) and human hemoglobin (pI 7.0) was used as markers for the pH gradient. A blank gel focused with each set of samples were used to measure the pH gradient at 25 °C. Gels were stained for protein with 0.1 ~ Coomassie Blue. Human placental lactogen was also detected by precipitation in the gel with the specific antibody according to the general procedure of Catsimpoolas [23] as modified by Powell et al. [24]. Following a 24 h incubation with anti-human placental lactogen serum (1:5000), the gels were washed extensively and then stained with 0.1 ~ Fast Green to disclose immunologically reactive bands. The isoelectric points of the bands were determined by reference to the pH gradient established along the gel. In some experiments, in order to insure that the sulfhydryl groups were all in the reduced state, human placental lactogen was first reduced with 10 -3 M dithiothreitol for 1 h at room temperature, then electrofocused in the presence of 10 -3, 5" 10 -3, 10 -2, or 5.10 -2 M p-chloromercuribenzoic acid, using pH 4-6 ampholyte in 4 ~o acrylamide gel. In order to unfold human placental lactogen molecules, electrofocusing was also carried out in the presence of 8 M urea under the same conditions. RESULTS Both standard preparations of human placental lactogen and all six preparations purified from individual placentas migrated as single bands on disc gel electrophoresis using 5 ~ polyacrylamide. However, when electrophoresis was carried out for 17 h at 4 °C, using a 7.5 ~ gel and a current of 2 mA/gel, the appearance of shoulders in the human placental lactogen peak suggested heterogeneity. This structural evidence of heterogeneity is clearer at the higher resolution obtainable on focusing. Initial studies by isoelectric focusing in sucrose gradients according to the method of Catsimpoolas [25] confirmed the heterogeneity of the standard placental lactogen samples but failed to provide sharp resolution because of precipitation of the concentrated protein bands at their isoelectric points~ Isofocusing was therefore carried out using polyacrylamide gels [22]. The commercial preparations resolved into six isoproteins with pI values of 5.0, 5.5, 5.8, 6.0, 6.1 and 6.2 at 24 °C. The density of individual bands varied considerably. Moreover, different standard preparations contained different proportions of the isoproteins (Fig. 1). In order to establish the stability of each isoprotein band, individual bands of the standard preparation were extracted and refocused separately (Fig. 2A). Under these conditions, the isoproteins migrated to the same isoelectric point as in the mixture. Fig. 2A also illustrates that applying two different quantities of human placental lactogen did not affect the positions of the bands. To eliminate the possibility that these isoproteins
335
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Fig. 1. Pattern of two standard commercial human placental lactogen samples (Nutritional Biochemicals Batch Nos. 3359 and 5501) and six human placental lactogen samples prepared from individual placentas that had been subjected to isofocusing. Six isoprotein bands with pI values of 5.0, 5.5, 5.8, 6.0, 6.1 and 6.2 are seen when read from bottom (lowest pI) to top (highest pI).
represent different conformations of the same molecule, isofocusing was carried out in the presence of 8 M urea to unfold the polypeptide chain. Fig. 2B shows that multiple bands are still evident in the presence of urea. A further test was applied to isofocus placental lactogen (previously treated with dithiothreitol) in the presence of different concentrations of p-chloromercuribenzoate (Fig. 2C). The persistence of multiple bands in these gels indicated that variation in disulfide bonding does not account for the observed heterogeneity. As an indication that the isoprotein bands represented immunologically related molecules, each was extracted and reacted against antibody prepared against the commercial placental lactogen sample by the Ouchterlony immunodiffusion method. Fig. 3 demonstrates the immunological identity of all isoprotein bands. If the original human placental lactogen sample had contained a mixture of structurally unrelated proteins, this uniformity of precipitation bands would not have been obtained. Human placental lactogen extracted from six full term placentas was also isofocused with the following results (Fig. 1). All samples contained several isoproteins corresponding in pI value to those in the pooled human placental lactogen sample. The number of bands identified varied from 3 to 6 for individual samples, and the densities of the different bands also varied from placenta to placenta. No repeating pattern could be discerned among the six samples. By comparison with the two commercial preparations, shown in Fig. 1, the constant presence of the isoproteins with pI values 5.0 and 5.5 is reflected in the predominance of these bands in the pooled preparation. Finally, the identity of the isoprotein bands was tested by reacting the resolved gel with antiserum to commercial placental lactogen. All bands staining for protein also reacted with the antibody. This indicates that the multiple bands in these individual preparations are not due to contaminating proteins but represent authentic isoforms of human placental lactogen.
336
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Fig. 2. (A) Isofocusing of human placental lactogen (Lederle, purified placental protein, 95 ~ pure), 5 ktg sample, to show effect of removing bands and refocusing them individually. Ampholyte (2 ~ , w/v) of range pH 4-6 was used in 4 ~ (w/v) acrylamide gel. The gels were staine~with 0.1 ~ Coomassie Blue after electrophoretic deampholytisation. Gels 1 and 2, native human placental tactogen (one at twice the loading level), showing bands at pI 6.1, 6.0, 5.8, 5.6, and 5.5 (from above downwards); Gel 3 : refocusing of pI 6.0 fraction; Gel 4, refocusing of pI 5.8 fraction; Gel 5, refocusing of pI 6.0 and 5.8 fractions; Gel 6, refocusing of pI 5.6 fraction; Gels 7 and 8, refocusing of pH 5.5 fraction. (B) lsofocusing of Lederle human placental lactogen with 8 M urea (track 1) compared with no urea in ampholyte (track 2). Bands occur at pI values of 6.1, 6.0, 5.6 and 5.5 (from above downwards). (C) Isofocusing of Lederle human placental lactogen (reduced with dithiothreitol) using pH 4.6 ampholyte in 4 ~ acrylamide gels in the presence of(l) 5.10 -2 (2) 10-2 (3) 5.10 -3 (4) 10 -3 M p-chloromercuribenzoate; sample 5 was untreated (control). The reduction of the disulfide bonds resulted in a shift in the bands with pI values of 6.1,6.0, 5.6 and 5.5 which nevertheless remain discrete. The gels were aligned using human hemoglobin as a marker.
DISCUSSION The preceding studies p r o v i d e evidence for m i c r o h e t e r o g e n e i t y o f h u m a n p l a c e n t a l lactogen in h u m a n placentas. A t t e m p t s to resolve isoproteins by polya c r y l a m i d e gel electrophoresis p r o v i d e d only suggestive evidence which m a y a c c o u n t for the lack o f identification o f multiple f o r m s in previous investigations. Schneider et al. [26] have indeed r e p o r t e d the presence o f a " b i g " h u m a n placental lactogen molecule in extracts o f h u m a n p l a c e n t a a n d serum following gel filtration t h r o u g h Sephadex G-100. O n p o l y a c r y l a m i d e electrophoresis o f big h u m a n placental lactogen in presence o f urea, two b a n d s were resolved, one o f which coincided with native h u m a n placental lactogen o f m o l e c u l a r weight o f 22 000. However, in o u r hands, isoelectric focusing p r o v i d e d the necessary sensitivity to o b t a i n several forms o f
337
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Fig. 3. Ouchterlony immunodiffusion of juxtaposed pI fractions 6.1, 6.0, 5.8, 5.6, and 5.5, obtained by isofocusing the commercial Lederle human placental lactogen sample. The human placental lactogen fractions were placed randomly in the wells. The center well contained anti-human placental lactogen serum. Different amounts of the isolated pI fractions were added to each set of wells, the largest amount being used for the center set. The complete identity of all samples is evidenced by the continuity of the precipitation lines. human placental lactogen. These appear to be authentic differing peptides of stable structure; since refocusing does not alter their pI values, the bands are not deleted by urea denaturation or treatment with p-chloromercuribenzoate and the radial immunodiffusion test indicates identity of antigenicity. Recently, Belleville et al. [12] have resolved human placental lactogen into two forms with isoelectric points of 4.6 and 4.7, which differ only in the degree of oxidation of intrachain disulfide groups. This finding differs from ours in the multiplicity of forms, isoelectric points obtained, and in the lack of conversion of one form to another by sulfhydryl reagents. Their use of lower voltage isofocusing for a shorter time may have lacked the necessary sensitivity to detect more bands. Other placental proteins have been shown to exhibit multiple forms. Thus, two major forms of placental alkaline phosphatase have been identified in a Caucasian population, and an additional form in a Negro population [27]. The two major forms appear to be interconvertible on prolonged storage [1]. Four forms of leucine aminopeptidase have been characterised [3] which differ in their sialic acid contents. Finally, Wiggert and Villee [2] have described five different tetramers of lactate dehydrogenase and two forms of malate dehydrogenase. The former enzyme consists of two types of subunits in different combinations. None of these multiple forms has been attributed to a difference in the primary structure of the polypeptide chain, which seems the most acceptable explanation for our findings with human placental lactogen. Although, multiple forms of human choriogonadotropin [6, 7] have been ascribed to differences in sialic acid content, a report attributes this to differences in the Nterminal sequence of the a-subunit [8]. The occurrence of multiple primary forms is known for human pituitary prolactin [13] and human growth hormone [14, 15] which are homologous with human placental lactogen [16]. Several explanations are possible to account for heterogeneity of the human placental lactogen peptide chain. Boime [28-30] has described the in vitro synthesis of a precursor which is cleaved to the shorter mature peptide hormone while still nascent on the ribosome. However, this larger precursor form has not been identified
338 in placental extracts [31]. It is o f course, possible t h a t cleavage o f a p r e c u r s o r f o r m is imprecise so t h a t forms with one o r m o r e a d d i t i o n a l a m i n o acids are p r o d u c e d . W e have, however, no k n o w l e d g e o f examples o f this for o t h e r proteins. I n contrast, studies o f p r i m a r y structure p r o v i d e a few instances in which p r o t e i n s f r o m the same tissue occur with sequence differences. F o r e x a m p l e c a r b o n i c a n h y d r a s e s B a n d C o f erythrocytes show 40 ~o o f the a m i n o acid sequences to differ [32] a n d the two serine d e h y d r a t a s e s o f r a t liver differ by two a m i n o acids [33]. It has also been shown that p r o t e i n isomers can arise f r o m p o s t - t r a n s l a t i o n a l events such as d e a m i d a t i o n [34, 35] a n d c a r b a m y l a t i o n [36]. Such p o s t - t r a n s l a t i o n a l changes also cause m i c r o h e t e r o geneity. Finally, a l t h o u g h we c a n n o t rule o u t d e g r a d a t i v e changes in the p l a c e n t a following its expulsion f r o m the uterus, the c o n s t a n c y o f the b a n d patterns o b t a i n e d indicates t h a t the v a r i o u s forms are n o t the r a n d o m p r o d u c t s o f d e g r a d a t i o n . ACKNOWLEDGEMENTS W e wish to t h a n k the nursing staff o f the B o s t o n H o s p i t a l for w o m e n (Lying-in division) for help in the collection o f p l a c e n t a s ; a n d Dr. James W. D r y s d a l e for assistance with the isoelectric focusing a n d for helpful c o m m e n t s in the p r e p a r a t i o n o f this manuscript.
REFERENCES 1 Ghosh, N. K. and Fishman, W. H. (1967) Fed. Proc. 26, 558 2 Wiggert, B. O. and Villee, C. A. (1964) J. Biol. Chem. 239, 444-451 3 Beckman, I., Boorling, B. and Christopolou, C. (1966) Acta Genet. Basel 16, 122-131 4 0 y a , M., Yoshino, M. and Asano, M. (1974) Experientia 30, 985-986 5 0 y a , M. (1974) Experientia 31, 1019-1020 6 Hamashige, S., Astor, M. A., Arguilla, E. R. and Van Thiel, D. H. (1967) J. Clin. Endocrinol. 27, 1690-1694 7 Bell, J. J., Canfield, R. E. and Sciarra, J. J. (1969) Endocrinology 84, 298-304 8 Bellisario, R., Carlsen, R. B. and Bahl, O. P. (1973) J. Biol. Chem. 248, 6796-6805 9 Belleville, F., Nabet, P. and Paysant, P. (1972) C.R. Soc. Biol. 166, 1060-1064 10 Belleville, F., Paysant, P. and Nabet, P. (1973) C.R. Soc. Biol. 167, 301-305 11 Belleville, F., Lasbennes, A., Nabet, P. and Paysant, P. (1974) C.R. Soc. Biol. 168, 1057-1062 12 Belleville, F., Peltier, A., Paysant, P. and Nabet, P. (1975) Eur. J. Biochem. 51,429-435 13 Ben-David, M. and Chrambach, A. (1974) Endocr. Res. Commun. 1, 193-210 14 Chrambach, A., Yadley, R. A., Ben David, M. and Rodbard, D. (1973) Endocrinology 93,848857 15 Yadley, R. A., Rodbard, D. and Chrambach, A. (1973) Endocrinology 93, 866-873 16 Niall, H. D., Hogan, M. L., Sauer, R., Rosenblum, I. Y. and Greenwood, S. C. (1971) Proc. Natl. Acad. Sci. U.S. 68, 866-869 17 Friesen, H. G. (1965) Endocrinology 76, 369-381 18 Kumai, A. (1970) Endocr. Jap. 17, 605-609 19 Ouchterlony, O. (1949) Acta Pathol. Microbiol. Scand. 26, 507-514 20 Ornstein, L. (1964) Ann. N.Y. Acad. Sci. 121,321-349 21 Davis, B. L. (1964) Ann. N.Y. Acad. Sci. 121,404-427 22 Righetti, P. G. and Drysdale, J. W. (1973) Ann. N.Y. Acad. Sci. 209, 163-186 23 Catsimpoolas, N. (1973) Ann. N.Y. Acad. Sci. 209, 65-80 24 Powell, L. W., Alpert, E., Isselbacher, K. J. and Drysdale, J. W. (1975) Br. J. Hematol. 30, 47-55 25 Catsimpoolas, N. (1971) Sep. Sci. 6, 435M42 26 Schneider, A. B., Kowlaski, K. and Sherwood, L. M. (1975) Endocrinology 97, 1364-1372 27 Boyer, S. H. (1961) Science 134, 1002-1004
339 28 29 30 31 32 33
Boime, I., Boguslawski, S. and Caine, J. (1975) Biochem. Biophys. Res. Commun. 62, 103-109 Boime, I., McWilliams, D., Szczesna, E. and Camel, M. (1976) J. Biol. Chem. 251,820-825 Szczesna, E. and Boime, I. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1179-1183 Friesen, H., Suwa, S. and Pone, P. (1969) Rec. Prog. Horm. Res. 25, 161-205 Goldstone, A., Konceny, P. and Koenig, H. (1971) FEBS Lett. 13, 68-71 Pitot, H. C., Iwasaki, Y., Inoue, H., Kasper, C. and Mohrenweiser, H. (1972) Gann Monogr. Cancer Res. 13, 191-204 34 Lai, C. Y., Chen, C. and Horecker, B. L. (1970) Biochem. Biophys. Res. Commun. 40, 461-468 35 Midelfort, C. F. and Mehler, A. H. (1972) Proc. Natl. Acad. Sci. U.S. 69, t816-1819 36 Johnson, R. W., Roberson, L. E. and Kenney, F. T. (1973) J. Biol. Chem. 248, 4521-4527
M I C R O H E T E R O G E N E I T Y OF H U M A N PLACENTAL LACTOGEN SHOWING D I F F E R E N T PATTERNS IN INDIVIDUALS
MEERA CHATTERJEE, EMIL M. LAGA, CHARLES C. MERRILL and H A M I S H N. M U N R O
Physiological Chemistry Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of" Technology, Cambridge, Mass. 02139 (U.S.A.) (Received December 21st, 1976)
SUMMARY
Samples of human placental lactogen, obtained either as a standard pooled preparation or prepared from individual placentas, were shown to migrate as a single band on acrylamide gel electrophoresis. When subjected to isoelectric focusing in polyacrylamide gels, the pooled sample was resolved into bands at pI values 5.0, 5.5, 5.8, 6.0, 6.1 and 6.2. Different batches of the standard pooled sample gave different proportions of each isoprotein species. Isolation and refocusing of individual bands did not alter the pI of each. Treatment with urea or with p-chloromercuribenzoate did not eliminate microheterogeneity seen on isofocusing, indicating that the observed heterogeneity is probably not due to conformational differences or to restriction of molecular shape by disulfide bonds. It was shown by immunodiffusion that all the isofocusing bands reacted similarly against a common antibody to human placental lactogen. When placental lactogen was extracted from individual full term human placentas, the same isoprotein bands were observed but their proportions varied markedly from one placenta to another, and not all bands were present. Thus human placental lactogen displays considerable microheterogeneity which varies with individual placentas.
INTRODUCTION
Heterogeneity among placental proteins has been described for alkaline phosphatase [1], malate and lactate dehydrogenases [2], leucine aminopeptidase [3-5], as well as the other major peptide hormone, human choriogonadotropin [6, 7]. Variation in the latter was thought to be due to differences in its sialic acid content, but differences in the amino acid sequence at the N-terminal end of the a-subunit have since been observed [8]. Belleville et al. [9-12] have characterised human placental lactogen by polyacrylamide gel electrophoresis, describing "slow" and "fast" bands. These were also seen on I polyacrylamide isofocusing and were ascribed to the degree of oxidation of disulfide interchain linkages. Thus only one primary structure was considered to be present. However, Chrambach and his colleagues have shown that human pituitary prolactin [13] and human growth hormone [14, 15], which display structural homology with human placental lactogen [16], show multiple isoproteins by iso-
333 electric focusing. This suggests that isofocusing of human placental lactogen may also reveal microheterogeneity. We have accordingly used isoelectric focusing to examine batch commercial preparations of pooled human placental lactogen and also human placental lactogen prepared from individual placentas. This demonstrated marked microheterogeneity which varied in pattern from one placenta to another, and which cannot be attributed to disulfide linkages. MATERIALS AND METHODS
Preparation of human placental lactogen. Human placental lactogen which had been extracted from pooled term placentas by the method of Friesen [17] and stated to be 95 ~ pure and homogeneous by polyacrylamide electrophoresis, was purchased from Nutritional Biochemicals, (Batch Numbers 3359 and 5501) and from Lederle ("Purified Placental Protein"). In addition, human placental lactogen was extracted by us from individual placentas obtained at normal full term deliveries. The extraction and purification of the peptide hormone followed Kumai's [18] modification of the original Friesen procedure, with substitution of DEAE-cellulose for ECTEOLAcellulose. Each fresh term placenta was homogenized and then extracted at pH 8.5 followed by adjustment of the supernatant fraction to pH 4.6. The supernatant after this treatment was brought to 50~o saturation with (NH4)2SO4 and the precipitate was harvested. Following dialysis to remove the ammonium sulfate, the solution was applied to a Sephadex G-100 column equilibrated with 0.1 M (NH4)2CO3. The retarded fraction was then applied to a DEAE-cellulose column and eluted with increasing concentrations of NH4HCO3 (0.05, 0.1 and 0.3 M). The 0.3 M fraction contained human placental lactogen, and was lyophilized. This procedure was monitored by radioimmunoassay with the Schwarz-Mann assay kit to identify the most active fractions for human placental lactogen activity. Later, the Ouchterlony immunodiffusion test using anti-human placental lactogen sera prepared in the laboratory was also employed. Anti-human placental lactogen serum. New Zealand White rabbits (2.5 kg) were immunized with the commercial human placental lactogen sample, by injecting 1 mg human placental lactogen mixed with 1 ml complete Freund's adjuvant weekly for 5 weeks. The serum harvested at the end of 5 weeks was found to have a high titer against human placental lactogen. This potent antiserum was used to check the stages in human placental lactogen purification from individual placentas by the Ouchterlony double immunodiffusion method [19] and for identifying the individual bands obtained on isofocusing. Electrophoresis of human placental lactogen. All preparations were subjected to electrophoresis in 5 ~o polyacrylamide gels according to the method of Ornstein [20] and Davis [21]. A continuous Tris/glycine buffer system (pH 8.3) was used and electrophoresis was carried out at 2 mA/gel for 7-8 h or 5 mA/gel for 5-6 h at 25 °C or at 2 mA/gel for 17 h at 4 °C. The gels were scanned by a Gilford recording spectrophotometer at 280 nm. Isoelectric focusing of human placental lactogen. The apparatus used (Metalloglass, Inc. Boston, Mass.) was identical to that of Righetti and Drysdale [22]. Polyacrylamide gels (4~, with 0.8 ~ bis) containing 1.5 ~ ampholyte (LKB Produkter, AB), pH 3-10, 4-6, or 5-7, were used. Sample loads ranged from 5 to 100 #g protein
334 per gel (3 X 100 mm). It was determined that separation of 20 #g human placental lactogen was optimum for protein staining and 5/~g for immunological detection. An initial current of 0.5 mA/gel was applied and voltage maintained at 200 V for 17 h. A circulating coolant, 5 ~o ethylene glycol, was used to maintain the temperature of the gels at 0-4 °C. The anolyte used was 0.01 M HaPO4 and the catholyte 0.02 M NaOH. After brief electrolysis (1 mA/tube for 10 min) to discharge persulfate, the samples were applied through the catholyte onto the top of the gel in a dense buffered solution ( 5 ~ ampholyte) titrated to the pH at which the proteins were in anionic form. Horse spleen ferritin (pl 4.2-4.6) and human hemoglobin (pI 7.0) was used as markers for the pH gradient. A blank gel focused with each set of samples were used to measure the pH gradient at 25 °C. Gels were stained for protein with 0.1 ~ Coomassie Blue. Human placental lactogen was also detected by precipitation in the gel with the specific antibody according to the general procedure of Catsimpoolas [23] as modified by Powell et al. [24]. Following a 24 h incubation with anti-human placental lactogen serum (1:5000), the gels were washed extensively and then stained with 0.1 ~ Fast Green to disclose immunologically reactive bands. The isoelectric points of the bands were determined by reference to the pH gradient established along the gel. In some experiments, in order to insure that the sulfhydryl groups were all in the reduced state, human placental lactogen was first reduced with 10 -3 M dithiothreitol for 1 h at room temperature, then electrofocused in the presence of 10 -3, 5" 10 -3, 10 -2, or 5.10 -2 M p-chloromercuribenzoic acid, using pH 4-6 ampholyte in 4 ~o acrylamide gel. In order to unfold human placental lactogen molecules, electrofocusing was also carried out in the presence of 8 M urea under the same conditions. RESULTS Both standard preparations of human placental lactogen and all six preparations purified from individual placentas migrated as single bands on disc gel electrophoresis using 5 ~ polyacrylamide. However, when electrophoresis was carried out for 17 h at 4 °C, using a 7.5 ~ gel and a current of 2 mA/gel, the appearance of shoulders in the human placental lactogen peak suggested heterogeneity. This structural evidence of heterogeneity is clearer at the higher resolution obtainable on focusing. Initial studies by isoelectric focusing in sucrose gradients according to the method of Catsimpoolas [25] confirmed the heterogeneity of the standard placental lactogen samples but failed to provide sharp resolution because of precipitation of the concentrated protein bands at their isoelectric points~ Isofocusing was therefore carried out using polyacrylamide gels [22]. The commercial preparations resolved into six isoproteins with pI values of 5.0, 5.5, 5.8, 6.0, 6.1 and 6.2 at 24 °C. The density of individual bands varied considerably. Moreover, different standard preparations contained different proportions of the isoproteins (Fig. 1). In order to establish the stability of each isoprotein band, individual bands of the standard preparation were extracted and refocused separately (Fig. 2A). Under these conditions, the isoproteins migrated to the same isoelectric point as in the mixture. Fig. 2A also illustrates that applying two different quantities of human placental lactogen did not affect the positions of the bands. To eliminate the possibility that these isoproteins
335
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represent different conformations of the same molecule, isofocusing was carried out in the presence of 8 M urea to unfold the polypeptide chain. Fig. 2B shows that multiple bands are still evident in the presence of urea. A further test was applied to isofocus placental lactogen (previously treated with dithiothreitol) in the presence of different concentrations of p-chloromercuribenzoate (Fig. 2C). The persistence of multiple bands in these gels indicated that variation in disulfide bonding does not account for the observed heterogeneity. As an indication that the isoprotein bands represented immunologically related molecules, each was extracted and reacted against antibody prepared against the commercial placental lactogen sample by the Ouchterlony immunodiffusion method. Fig. 3 demonstrates the immunological identity of all isoprotein bands. If the original human placental lactogen sample had contained a mixture of structurally unrelated proteins, this uniformity of precipitation bands would not have been obtained. Human placental lactogen extracted from six full term placentas was also isofocused with the following results (Fig. 1). All samples contained several isoproteins corresponding in pI value to those in the pooled human placental lactogen sample. The number of bands identified varied from 3 to 6 for individual samples, and the densities of the different bands also varied from placenta to placenta. No repeating pattern could be discerned among the six samples. By comparison with the two commercial preparations, shown in Fig. 1, the constant presence of the isoproteins with pI values 5.0 and 5.5 is reflected in the predominance of these bands in the pooled preparation. Finally, the identity of the isoprotein bands was tested by reacting the resolved gel with antiserum to commercial placental lactogen. All bands staining for protein also reacted with the antibody. This indicates that the multiple bands in these individual preparations are not due to contaminating proteins but represent authentic isoforms of human placental lactogen.
336
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Fig. 2. (A) Isofocusing of human placental lactogen (Lederle, purified placental protein, 95 ~ pure), 5 ktg sample, to show effect of removing bands and refocusing them individually. Ampholyte (2 ~ , w/v) of range pH 4-6 was used in 4 ~ (w/v) acrylamide gel. The gels were staine~with 0.1 ~ Coomassie Blue after electrophoretic deampholytisation. Gels 1 and 2, native human placental tactogen (one at twice the loading level), showing bands at pI 6.1, 6.0, 5.8, 5.6, and 5.5 (from above downwards); Gel 3 : refocusing of pI 6.0 fraction; Gel 4, refocusing of pI 5.8 fraction; Gel 5, refocusing of pI 6.0 and 5.8 fractions; Gel 6, refocusing of pI 5.6 fraction; Gels 7 and 8, refocusing of pH 5.5 fraction. (B) lsofocusing of Lederle human placental lactogen with 8 M urea (track 1) compared with no urea in ampholyte (track 2). Bands occur at pI values of 6.1, 6.0, 5.6 and 5.5 (from above downwards). (C) Isofocusing of Lederle human placental lactogen (reduced with dithiothreitol) using pH 4.6 ampholyte in 4 ~ acrylamide gels in the presence of(l) 5.10 -2 (2) 10-2 (3) 5.10 -3 (4) 10 -3 M p-chloromercuribenzoate; sample 5 was untreated (control). The reduction of the disulfide bonds resulted in a shift in the bands with pI values of 6.1,6.0, 5.6 and 5.5 which nevertheless remain discrete. The gels were aligned using human hemoglobin as a marker.
DISCUSSION The preceding studies p r o v i d e evidence for m i c r o h e t e r o g e n e i t y o f h u m a n p l a c e n t a l lactogen in h u m a n placentas. A t t e m p t s to resolve isoproteins by polya c r y l a m i d e gel electrophoresis p r o v i d e d only suggestive evidence which m a y a c c o u n t for the lack o f identification o f multiple f o r m s in previous investigations. Schneider et al. [26] have indeed r e p o r t e d the presence o f a " b i g " h u m a n placental lactogen molecule in extracts o f h u m a n p l a c e n t a a n d serum following gel filtration t h r o u g h Sephadex G-100. O n p o l y a c r y l a m i d e electrophoresis o f big h u m a n placental lactogen in presence o f urea, two b a n d s were resolved, one o f which coincided with native h u m a n placental lactogen o f m o l e c u l a r weight o f 22 000. However, in o u r hands, isoelectric focusing p r o v i d e d the necessary sensitivity to o b t a i n several forms o f
337
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Fig. 3. Ouchterlony immunodiffusion of juxtaposed pI fractions 6.1, 6.0, 5.8, 5.6, and 5.5, obtained by isofocusing the commercial Lederle human placental lactogen sample. The human placental lactogen fractions were placed randomly in the wells. The center well contained anti-human placental lactogen serum. Different amounts of the isolated pI fractions were added to each set of wells, the largest amount being used for the center set. The complete identity of all samples is evidenced by the continuity of the precipitation lines. human placental lactogen. These appear to be authentic differing peptides of stable structure; since refocusing does not alter their pI values, the bands are not deleted by urea denaturation or treatment with p-chloromercuribenzoate and the radial immunodiffusion test indicates identity of antigenicity. Recently, Belleville et al. [12] have resolved human placental lactogen into two forms with isoelectric points of 4.6 and 4.7, which differ only in the degree of oxidation of intrachain disulfide groups. This finding differs from ours in the multiplicity of forms, isoelectric points obtained, and in the lack of conversion of one form to another by sulfhydryl reagents. Their use of lower voltage isofocusing for a shorter time may have lacked the necessary sensitivity to detect more bands. Other placental proteins have been shown to exhibit multiple forms. Thus, two major forms of placental alkaline phosphatase have been identified in a Caucasian population, and an additional form in a Negro population [27]. The two major forms appear to be interconvertible on prolonged storage [1]. Four forms of leucine aminopeptidase have been characterised [3] which differ in their sialic acid contents. Finally, Wiggert and Villee [2] have described five different tetramers of lactate dehydrogenase and two forms of malate dehydrogenase. The former enzyme consists of two types of subunits in different combinations. None of these multiple forms has been attributed to a difference in the primary structure of the polypeptide chain, which seems the most acceptable explanation for our findings with human placental lactogen. Although, multiple forms of human choriogonadotropin [6, 7] have been ascribed to differences in sialic acid content, a report attributes this to differences in the Nterminal sequence of the a-subunit [8]. The occurrence of multiple primary forms is known for human pituitary prolactin [13] and human growth hormone [14, 15] which are homologous with human placental lactogen [16]. Several explanations are possible to account for heterogeneity of the human placental lactogen peptide chain. Boime [28-30] has described the in vitro synthesis of a precursor which is cleaved to the shorter mature peptide hormone while still nascent on the ribosome. However, this larger precursor form has not been identified
338 in placental extracts [31]. It is o f course, possible t h a t cleavage o f a p r e c u r s o r f o r m is imprecise so t h a t forms with one o r m o r e a d d i t i o n a l a m i n o acids are p r o d u c e d . W e have, however, no k n o w l e d g e o f examples o f this for o t h e r proteins. I n contrast, studies o f p r i m a r y structure p r o v i d e a few instances in which p r o t e i n s f r o m the same tissue occur with sequence differences. F o r e x a m p l e c a r b o n i c a n h y d r a s e s B a n d C o f erythrocytes show 40 ~o o f the a m i n o acid sequences to differ [32] a n d the two serine d e h y d r a t a s e s o f r a t liver differ by two a m i n o acids [33]. It has also been shown that p r o t e i n isomers can arise f r o m p o s t - t r a n s l a t i o n a l events such as d e a m i d a t i o n [34, 35] a n d c a r b a m y l a t i o n [36]. Such p o s t - t r a n s l a t i o n a l changes also cause m i c r o h e t e r o geneity. Finally, a l t h o u g h we c a n n o t rule o u t d e g r a d a t i v e changes in the p l a c e n t a following its expulsion f r o m the uterus, the c o n s t a n c y o f the b a n d patterns o b t a i n e d indicates t h a t the v a r i o u s forms are n o t the r a n d o m p r o d u c t s o f d e g r a d a t i o n . ACKNOWLEDGEMENTS W e wish to t h a n k the nursing staff o f the B o s t o n H o s p i t a l for w o m e n (Lying-in division) for help in the collection o f p l a c e n t a s ; a n d Dr. James W. D r y s d a l e for assistance with the isoelectric focusing a n d for helpful c o m m e n t s in the p r e p a r a t i o n o f this manuscript.
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Boime, I., Boguslawski, S. and Caine, J. (1975) Biochem. Biophys. Res. Commun. 62, 103-109 Boime, I., McWilliams, D., Szczesna, E. and Camel, M. (1976) J. Biol. Chem. 251,820-825 Szczesna, E. and Boime, I. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1179-1183 Friesen, H., Suwa, S. and Pone, P. (1969) Rec. Prog. Horm. Res. 25, 161-205 Goldstone, A., Konceny, P. and Koenig, H. (1971) FEBS Lett. 13, 68-71 Pitot, H. C., Iwasaki, Y., Inoue, H., Kasper, C. and Mohrenweiser, H. (1972) Gann Monogr. Cancer Res. 13, 191-204 34 Lai, C. Y., Chen, C. and Horecker, B. L. (1970) Biochem. Biophys. Res. Commun. 40, 461-468 35 Midelfort, C. F. and Mehler, A. H. (1972) Proc. Natl. Acad. Sci. U.S. 69, t816-1819 36 Johnson, R. W., Roberson, L. E. and Kenney, F. T. (1973) J. Biol. Chem. 248, 4521-4527
