The Purification and Characterization of Rat Placental Lactogen MAY C. ROBERTSON 1 AND HENRY G. FRIESEN Department of Physiology, Faculty of Medicine, University of Manitoba Winnipeg, Canada ABSTRACT. A method has been developed for the purification of rat placental lactogen employing a specific radioreceptor assay (RRA). The method involves precipitation with ammonium sulfate, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative isoelectric focusing. The isolation procedure results in a 1300-fold purification and a 10% yield of rat placental lactogen. Potency estimates

Manitoba,

by RRA indicate that the purified hormone is 41% as active as the ovine prolactin standard (25 I U/mg), but 169% as active as the NIH human placental lactogen preparation. Polyacrylamide gel electrophoresis at pH 8.9 and analytical isoelectric focusing of the rat placental lactogen reveal 2 major and 2 minor components, all of which are active in the RRA. (Endocrinology 97: 621, 1975)

T

Purification

HE presence of a mammotropic and/or luteotropic substance in rat placenta which is important for the maintenance of pregnancy has been well established (1-4). Using a radioreceptor assay (RRA) for lactogenic hormones, we were able to determine the concentration of placental lactogen in serum and placental tissue at different periods of gestation in the rat (5). These studies confirmed the existence of maximal serum concentrations of lactogen on day 12 of pregnancy, as reported by Matthies (6). In placental tissue, the peak concentration of lactogen was found between days 15 and 17. The present report describes a method for the isolation and characterization of lactogenic hormone from late pregnant rat placentas.

All steps were carried out at 4 C unless otherwise specified. 1) Extraction and ammonium sulfate precipitation Placentas from 50-60 rats (160 g) were homogenized with a Polytron homogenizer at top speed for 1 min in 800 ml of a mixture composed of equal volumes of 0.1M ammonium bicarbonate and 0.1N ammonium hydroxide (pH 9.2) (5 vol/ g of tissue). The homogenate was stirred for 2-4 h at 4 C and 40 ml of 0.5M benzamidine in water was added to give a final concentration of 20 mM. The homogenate was centrifuged at 15,000 x g for 30 min and the supernatant was centrifuged at 100,000 x g for a further 2 - 3 h. To the 100,000 x g supernatant, solid ammonium sulfate was added slowly, with stirring, to achieve 60% saturation (39 g/100 ml). After stirring the suspension for one hour, the precipitate was allowed to settle overnight and was collected by centrifugation at 27,000 x g for 30 min.

Materials and Methods Tissue source Rats at days 17-19 of pregnancy were sacrificed by guillotine and fetal placentas were collected immediately, dissected free of membranes, blotted to remove excess blood and stored frozen at - 2 0 C until use. Usually, 2 - 3 g of placental tissue was collected from one pregnant rat. Received October 29, 1974. 1 Present address: Max-Planck-Institut fur Experimentelle Medizin, Abteilung Molekulare Genetik, 3400 Gottingen, Hermann-Rein-Strasse 3, Germany.

2) Sephadex gel filtration The 60% ammonium sulfate precipitate was extracted with approximately 200 ml of 0.05M ammonium bicarbonate (pH 8.2). The solution was centrifuged at 15,000 x g for 10 min and any remaining precipitate generally dissolved readily with an additional volume of 0.05M ammonium bicarbonate. The two extracts were combined and applied to a Sephadex G-100 column (8.0 x 80 cm) equilibrated with 0.05M ammonium bicarbonate, pH 8.2. The fractions collected were

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622

ROBERTSON AND FRIESEN

analyzed spectrophotometrically at 280 and 260 nm and by RRA for placental lactogen. Appropriate fractions were pooled for chromatography on DEAE cellulose. 3) DEAE-cellulose ion exchange chromatography The pool from the Sephadex column containing placental lactogen was diluted twofold and applied to a column (2.2 x 27 cm) of diethylaminoethyl cellulose. (Whatman DE32) previously equilibrated with 0.05M ammonium bicarbonate, pH 8.0. After washing the column with 100 ml of starting buffer, elution was carried out with a gradient formed from 500 ml each of 0.05 and 0.2M ammonium bicarbonate, followed by a second gradient from 0.2M to 1.0M ammonium bicarbonate. 4) CM-Sephadex ion exchange

chromatography

In order to change the counter ion from sodium to ammonium, about 10 g of carboxymethyl Sephadex (Pharmacia C-25) was allowed to swell overnight in 1M ammonium acetate. The gel was washed several times to reduce the ionic strength to 0.01M before pouring the column. From two DEAE cellulose columns, fractions which were rich in placental lactogen were pooled and dialysed overnight against 0.01M ammonium bicarbonate. The pH of the dialysate was adjusted to 6.2 with dilute formic acid and applied to a column (2 x 14 cm) of CM-Sephadex equilibrated in 0.05M ammonium formate, pH 6.2. The column was washed with 250 ml of 0.1M ammonium formate before elution was carried out employing a pH gradient using equal volumes (350 ml) of 0.10M ammonium formate (pH 6.2) and 0.2M ammonium bicarbonate (pH 8.2). A second gradient, formed from 250 ml 0.2M ammonium bicarbonate (pH 8.2) and 250 ml 0.5M ammonium bicarbonate (pH 8.5), was also applied. In some cases, as judged by the strong red color, some hemoglobin was also carried through the isolation procedure in the same fractions as the hormone. An additional gel filtration on Sephadex G-100 (3.0 x 65 cm) was used to separate this contaminant from the hormone before proceeding to the isoelectric focusing step.

I-:>RI •

1975

Vol 97 • No 3

with the cathode placed in the upper electrode position. The fractions containing placental lactogen from the CM-Sephadex column were dialysed and concentrated to a volume less than 50 ml in an Amicon cell with a UM-10 membrane. Column reagents and samples were prepared in an LKB 8121 gradient mixer and were applied at the rate of 1-2 ml/min with a peristaltic pump (LKB). Isoelectric focusing was allowed to proceed until a constant current was achieved (2024 h). The column contents were emptied by pumping water into the top of the column at a flow rate of 2 ml/min and collecting 2 ml fractions from the outlet at the bottom of the column. Protein concentrations were monitored by absorbance at 280 nm, and placenta] lactogen content by RRA. To separate ampholytes from proteins, tractions rich in placental lactogen were pooled and applied to a Sephadex G-100 column (3.0 x 65 cm) equilibrated with 0.05M ammonium bicarbonate. The ampholytes were monitored by their absorbance at 260 nm. Protein estimation Protein concentrations were estimated spectrophotometrically measuring absorbance at 280 and 260 nm (8). The protein content of the original homogenate and of the pools obtained at different stages of fractionation was determined by the method of Lowry et al. (9) using bovine serum albumin as standard. Radioreceptor assay The rabbit mammary gland receptor assay described previously (10,11) was used to monitor the concentration of placental lactogen, and the rabbit liver receptor assay (12) was used to measure growth hormone-like material. Hormone preparations and

radioimmunoassays

Rat pituitary prolactin, growth hormone and human placental lactogen preparations were generously provided by the NIAMDD program of the NIH. The radioimmunoassays for rat prolactin and growth hormone were performed according to the instructions supplied.

5) Isoelectric focusing The technique used was that recommended in the 8100 Ampholine instruction manual (7), with an isoelectric-focusing column of 110 ml capacity (LKB Ins., Broma, Sweden), carrier ampholytes with a pH range from 3.5 to 10, and

Characterization 1) Gel electrophoresis. Polyacrylamide gel electrophoresis was carried out as described by Davis (13) and Reisfeld (14). Gel electrophoresis in sodium dodecyl sulfate, SDS, was performed

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PURIFICATION OF RAT PLACENTAL LACTOGEN according to the method of Weber and Osborn (15). The gel concentrations have been indicated in the legend to the figures. 2) Analytical gel isoelectric focusing. Isoelectric focusing was performed using a LKB 2117 multiphor apparatus employing ampholytes which provided a pH range 3.5 to 9.5 with a 6% concentration of acrylamide + bisacrylamide (%T) and a cross linking of 2.5% (%C). Isoelectric focusing was usually continued for 5-8 h or until a sample of cytochrome C which was applied to the gel near both electrodes had migrated to form a single band near the cathode. In general, after disc gel acrylamide electrophoresis and thin layer isoelectric focusing of proteins in test samples, one disc gel or longitudinal gel slice was stained for protein while another one was serially divided into 2 - 3 mm segments. Each slice was eluted overnight in 1 to 2 ml of O.lM Tris-HCl buffer, pH 7.6, containing 0.1% (wt/vol) BSA, and the eluates subsequently analyzed by RRA.

Results Placentas from rats at days 17-19 of pregnancy were chosen for extraction because of their high content of placental lactogen. Placentas from rats closer to term (days 20 and 21) were avoided as initial studies had shown that these placentas contain substances which tend to clog the Sephadex columns. As shown in Fig. 1, over twice as much

FIG. 1. Quantity of rat placental lactogen extracted as a function of pH and time. One gram of rat placental tissue was homogenized in either 10 ml of equal volumes of 0 . 1 M ammonium bicarbonate and O.lN ammonium hydroxide, pH 9.2, or 10 ml of equal volumes of 0.1M ammonium acetate and O.lN acetic acid, pH 4.6. The homogenate was stirred at 4 C. Aliquots were removed at the times indicated and stored frozen until assayed. The abscissa is the time of removal of aliquots from the extraction mixture.

623

hormone was extracted in 4 h at 4 C and pH 9.2 than at pH 4.6. An extraction period of 2-4 h was sufficient to solubilize most of the hormone. Benzamidine, a reversible inhibitor of trypsin and plasmin, was added to reduce subsequent enzymatic degradation of placental lactogen. Figure 2 shows the distribution of protein and placental lactogen after gel filtration of the ammonium sulfate precipitate on Sephadex G-100. Most of the material which reacted in the mammary gland receptor assay emerged in fractions with an elution volume which is 1.9-2.4 times greater than the void volume of the column. Figure 3 shows the elution pattern when fractions which contained placental lactogen from the G-100 column were pooled and subjected to DEAE cellulose ion exchange chromatography. About 85% of the placental lactogen eluted between 0.1 and 0.15M ammonium bicarbonate. Increasing the molarity to 1.0M ammonium bicarbonate failed to remove additional placental lactogen. An aliquot of thei fraction which contained placental lactogen after DEAE cellulose chromatography was applied to a CM-Sephadex column for additional purification. As shown in Fig. 4, placental lactogen was eluted when the pH was increased from 6.2 to 8.0 upon the addition of 0.2M ammonium bicarbonate.

30 PH

9.2

25

20 O 2

I 15 E

PH4.6

J 2

4

6

L 10

12

24

48

TIME (HOURS)

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624

FIG. 2. Gel filtration of the 60% ammonium sulfate precipitate on Sephadex G-100 column (8.0 x 80 cm) using 0.05M am-

monium bicarbonate as buffer. The concentration of rat placental lactogen is expressed as equivalents of ovine prolactin.

1000

1500

2000

2500

4000

3500

3000

ELUTION VOLUME

achieved. When sufficient placental tissue was available, appropriate fractions from two DEAE chromatography columns were often pooled prior to application on the CMSephadex column in order to reduce nonspecific adsorption of placental lactogen. A total of 760 ixg purified rat placental lactogen was obtained from 350 g of rat placental tissue which contained 7.8 mg of placental lactogen, representing a recovery of 9.7%. The protein pattern upon gel isoelectric focusing at three of the purification steps is shown in Fig. 7. Preparations of NIH rat growth hormone, rat prolactin and human placental lactogen also displayed a number

When the fractions which contained pla-

cental lactogen from the CM-Sephadex column were subjected to isoelectric focusing, three major protein peaks were resolved (Fig. 5). Placental lactogen was concentrated in fractions with a pH between 6 and 7 and subsequent gel filtration of this pool (Fig. 6) showed that it contained predominantly rat placental lactogen. A summary of the purification procedure is presented in Table 1. The recovery of placental lactogen exceeded 70% at each step except in the ion exchange chromatography on DEAE cellulose and CM-Sephadex where recoveries of 50-60% were

-1.0 1

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ing the volume beginning with the linear gradient. The broken line indicates

FIG. 3. DEAE-cellulose chromatography of lactogen-rich fraction obtained from the Sephadex column whose elution pattern is shown in Fig. 2. The sample was applied in 0.05M ammonium bicarbonate. The first arrow indicates elution with 0.05M ammonium bicarbonate after the sample has been applied. The second arrow indicates the start of the first gradient from 0.05 to 0.2M ammonium bicarbonate, and the third arrow that of the start of the gradient formed from 0.2M to 1.0M ammonium bicarbonate. The abscissa is interrupted, the first part indicating the sample volume applied; and the second component indicatthe estimated salt concentration.

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PURIFICATION OF RAT PLACENTAL LACTOGEN

FIG. 4. CM-Sephadex chromatography of the lactogen-rich fraction from the DEAE-cellulose column (Fig. 3). Prior to application, the sample had been dialyzed against 0.01M ammonium bicarbonate and the pH adjusted to 6.2 with formic acid. The first arrow indicates the start of the elution with 0.1M ammonium formate pH 6.2 after the sample has been applied. The second arrow designates the start of the first gradient from 0.1M ammonium formate to 0.2M ammonium bicarbonate, and the third arrow that of the second gradient to 0.5M ammonium bicarbonate (pH 8.5).

400

800

1000

100

250

625

1000

500

ELUTION VOLUME (ML)

of bands when analyzed by this sensitive technique. Figure 8 shows the electrophoretic patterns of purified rat placental lactogen. Upon acrylamide gel electrophoresis at an alkaline pH (Fig. 8A), and upon isoelectric focusing (Fig. 8C), four components were observed. To determine the distribution of activity in the gel, individual gels were cut into successive 2mm segments, eluted, and assayed. Lactogenic activity measured by

RRA was found only in the eluates from the gel segments which corresponded to the bands seen on the gel. In addition, when serial dilutions of the eluates from these segments were made, the response curves observed in the receptor assay showed complete parallelism to that of the ovine prolactin standard. When electrophoresis was carried out at pH 4.3 acrylamide gel (Fig. 8B), only one component with an Rf of 0.5

FIG. 5. The elution profile of the lactogenic material from the CM-Sephadex column (Fig. 4) after isoelectric focusing in a sucrose gradient containing 1% carrier ampholytes in the pH 3-10 range. The closed circles indicate the pH of the fractions.

15

20

25

30

35

40

45

50

55

60

FRACTION NUMBER (2mlAube)

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ROBERTSON AND FRIESEN

626

FlG. 6. Gel chromatography on a Sephadex G-100 column (3.0 x 65 cm) of fractions 27 to 33 from the isoelectric focusing column of Fig. 5. The first arrow indicates the void volume of the column. The material eluting between the second and third arrows was pooled, concentrated and used for subsequent characterization procedures. Electrophoresis patterns are from 10% gels run in SDS at pH 7.2. The "a" pattern represents the material eluting between 280 and 300 ml, while the " b " pattern is the purified rat placental lactogen which eluted between 320 and 355 ml.

T

150

200

250

300

350

400

ELUTION VOLUME

450

(ml)

was observed. Upon electrophoresis in SDS gels, one major protein band with an Rf = 0.56, corresponding to a molecular weight of 22,000, was observed. In addition, there was a minor band with an Rf= 0.76 and an estimated weight of 12,000 (Fig. 6, gel b). When the purified rat placental lactogen was assayed at a concentration of 60 /u,g/ml by the appropriate radioimmunoassays, no evidence of contamination by rat pituitary growth hormone or rat prolactin was detected. With the kits supplied by NIAMDD,

the sensitivity of the assay for rat pituitary GH and PRL was 5 ng/ml and 2.5 ng/ml, respectively. When assayed in the rabbit mammary gland receptor assay using ovine [125I]iodoprolactin, the purified rat placental lactogen was 41% as effective as ovine prolactin (25.6 IU/mg) in competing for binding sites on the mammary gland receptor (Fig. 9A). A preparation of NIH-hPL was only 25% as effective as oPRL in this assay. Since the ability of a hormone to compete with [I25I]iodo-

TABLE 1. Recovery and yield of rat placental lactogen during purification Rat Placental Lactogen

Purification step

Protein1 mg

mg

% of total protein

% recovery

Purification factor

100,000 x g supernatant

8,840

3.90

0.044

100

1

60% ammonium sulfate precipitate

100

1.2

100

8.9

7,900

4.11

0.052

Sephadex G-100

922

3.90

0.394

DEAE-cellulose

360

2.36

0.655

60.5

14.9

CM-Sephadex*



2.56



32.8



Sephadex G-100 Isoelectric focusing Sephadex G-100

1.50

14.2

19.2

323

2

1.44

27.2

18.5

618

3

0.76

58.8

9.7

1,336

10.6 5.3

1.3

* Materials from two DEAE-cellulose columns were combined and applied to the CM-Sephadex column. 1 Protein estimation by the method of Lowry et al. (8) except when otherwise noted. 2 Protein estimation based on absorbance at 280 nm. 3 Dry weight.

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pH FIG. 7. Protein patterns on analytical gel isoelectric focusing over the pH range 3.5-9.5 of fractions taken at three stages of purification of rat placental lactogen A) Sephadex G-100(230 /xg) 1st gel filtration; B) DEAEcellulose chromatography (140 /xg); and C) CM-Sephadex chromatography (110 /xg). The other fractions are D) NIH-Rat Prolactin 80 (/xg); E) NIH-Rat Growth Hormone (75 /xg); and F) NIH-hPL (150 /xg). The samples were applied at the wicks at the bottom of the gel near the cathode. The final pH of the gel in this region is 9.5, and at the anode at the top of the gel is 3.0. The pH gradient of this gel was similar to that shown in Fig. 8C.

+ve

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origin

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3000

R: 12.

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FIG. 8. Gel electrophoresis and isoelectric focusing patterns of the purified rat placental lactogen. A) Polyacrylamide gel electrophoresis in 7.5% gel at pH 8.9. B) Polyacrylamide gel electrophoresis in 7.5% gel at pH 4.3. C) Analytical gel isoelectric focusing. The anode was placed at segment 22 while the cathode was on segment 1. Eluates of gel segments were assayed and the distribution of rat placental lactogen was determined by receptor assay. In each case, a second disc gel or longitudinal slice of the gel was stained for protein. "A" and "R" in each panel refer to the amount of placental lactogen which was applied to the gel and which was recovered from gel eluates, respectively (determined by RRA). In panel A, 2 peaks of placental lactogen are shown which were coincident with the 2 major bands seen after staining another disc gel run in a similar manner. In panel B, only a single peak of placental lactogen was observed coincident with the major protein band. In panel C, one can see several protein bands clearly separated. The rPL activity was found in eluates of the gel obtained from this region.

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ROBERTSON AND FRIESEN

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Enclo • 1975 Vol 97 • No 3

10 FIG. 9. Displacement curves for rat and human placental lactogen in radioreceptor assays using particulate membrane fractions of rabbit mammary gland and liver. A) Upper Panel: Rabbit mammary gland membranes (prolactin receptor assay) were incubated with ovine [125I]iodoprolactin in the presence of increasing concentrations of 3 hormones. The ordinate represents the displacement of ovine [125I]-iodoPRL bound to membrane binding sites in the presence of sheep prolactin (oPRL), rat placental lactogen (rPL) and human placental lactogen (hPL). When no hormones were added, approximately 15% of the ovine [125I]-iodoPRL in the incubation medium (100,000 cpm) was bound to the membranes. B) Lower Panel: Rabbit liver membranes (growth hormone receptor assay) were incubated with ovine [125I]iodoGH in the presence of increasing concentrations of bGH, hPL and rPL. In the absence of any of the hormones, 20% of the bovine [125I]iodoGH (100,000 cpm) was bound to the membranes.

20 CN

30

o z UJ

u

5

hPL

oPRL

40 50 60

Q.

70 80 X

O I CN

o z LLJ

u

3 a.

10

100

1000

10,000

HORMONE CONCENTRATION ng/ml

PRL in the rabbit mammary gland receptor assay can be related to the biological potency of the hormone (10), the preparation of rat placental lactogen would appear to be a more effective lactogenic hormone than the NIH-hPL preparation. When tested in a growth hormone receptor assay using rabbit liver membranes, rat placental lactogen showed minimal growth hormone-like activity (Fig. 9B). With this assay, apparent growth hormone-like activity has been detected in late pregnant rat

serum, but its level in rat placental tissue was less than 1% of the level of rat placental lactogen. Discussion Placental lactogen has been prepared in a highly purified form from late pregnant rat placentas. Purification of rat placental lactogen in quantities sufficient for complete chemical studies has not yet been attempted because of the low yield of the hormone and the lack of an adequate number of rat placen-

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PURIFICATION OF RAT PLACENTAL LACTOGEN tas. When one examines the degree of purification achieved at each stage of the isolation scheme (Table 1), it appears that a better recovery might be attained if the DEAE cellulose step were omitted, as a substantial loss of material occurs at this step with little gain in purification. The molecular weight of the isolated hormone was estimated to be 22,000 by electrophoresis in SDS-acrylamide gels using appropriate protein standards, and 18,000 when determined by gel filtration on a calibrated Sephadex G-100 column. These values are similar to those found for other placental lactogens (human and monkey) (16,17). The multiple protein components detected upon polyacrylamide electrophoresis in alkaline gel, and on thin layer isoelectric focusing suggest that rat placental lactogen may show heterogeneity similar to that reported for human growth hormone (18) and human and monkey placental lactogens (17). To clarify the differences which give rise to the polymorphism of either rat placental lactogen or pituitary hGH, it would, of course, be necessary to isolate and sequence each component. This, of course, has not been done for either hGH or placental lactogen. Based on the RRA results, the purity of rat placental lactogen was determined to be 60%. However, the purity of the preparation may be underestimated, since our estimate assumes that placental lactogens will compete on an equal basis with ovine prolactin in this assay. However, if one accepts that the purity of the NIH-hPL preparation is 90%, this assumption does not appear to be valid. With the availability of purified rat placental lactogen, a specific and sensitive radioimmunoassay can be developed which should prove very helpful in delineating the heterogeneity of rat placental lactogen (5) and also in defining the physiological

629

role of rat placental hormone during pregnancy. If sufficient quantities of the hormone can be obtained, comparative studies of the chemistry of rat placental lactogen and other lactogenic hormones should be feasible. Acknowledgments The technical assistance of Mrs. Gloria Balassu and the secretarial help of Mrs. Ruth Rannie are gratefully acknowledged. We thank Mr. Jeffrey Harris for preparing the figures. This research was supported by the Medical Research Council of Canada and a US Public Health Service Child Health and Human Development grant.

References 1. Astwood, E. B., and R. O. Greep, Proc Soc Exp Biol Med 38: 713, 1938. 2. Lyons, W. R., Anat Rec 88: 446, 1944. 3. Ray, E. W., S. C. Averill, W. R. Lyons, and R. E. Johnson, Endocrinology 56: 359, 1955. 4. Matthies, D. L. In Josimovich, J., M. Reynolds, and E. Cobo (eds.), Lactogenic Hormones, Fetal Nutrition and Lactation, Wiley, New York, 1974, p. 297. 5. Kelly, P. A., R. P. C. Shiu, M. C. Robertson, and H. G. Friesen, Fed Proc 32: 213, 1973. 6. Matthies, D. L., Anat Rec 159: 55, 1967. 7. LKB 8100 Ampholine Electrofocusing Equipment Instruction Manual, LKB-Produkter AB, S-161 25 Bromma 1, Sweden. 8. Warburg, D., and W. Christian, Biochem Z 310: 384, 1942. 9. Lowry, O. H., N. J. Rosebrough, A. L. Fair, and R. J. RandallJ Biol Chem 193: 265, 1951. 10. Shiu, R. P. C , P. A. Kelly, and H. G. Friesen, Science 180: 968, 1973. 11. , and H. G. Friesen, Biochem J 140: 301, 1974. 12. Tsushima, T., and H. G. Friesen, J Clin Endocrinol Metab 37: 334, 1973. 13. Davis, B. J.,Ann N Y Acad Sci 121: 404, 1964. 14. Reisfeld, R. A., U. J. Lewis, and D. E. Williams, Nature 195: 281, 1972. 15. Weber, K., and M. OsbornJ Biol Chem 244: 4406, 1969. 16. Friesen, H., Endocrinology 76: 369, 1965. 17. Shome, B., and H. G. Friesen, Endocrinology 89: 631, 1971. 18. Chrambach, A., R. A. Yadley, M. Ben-David, and D. Rodbard, Endocrinology 93: 848, 1973.

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The purification and characterization of rat placental lactogen.

A method has been developed for the purification of rat placental lactogen employing a specific radioreceptor assay (RRA). The method involves precipi...
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