The purification and characterization of ovine placental lactogen.

The Purification and Characterization of Ovine Placental Lactogen JOHN S. D. CHAN,1 H. A. ROBERTSON,2 AND H. G. FRIESEN 1 Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada, and Animal Research Institute, Ottawa, Ontario, Canada bovine growth hormone (bGH) and ovine prolactin (oPRL) standards and the ratio of GH-activity to PRL-activity of oPL is 1:2. In a body weight gain assay using hypophysectomized rats, oPL has a growthpromoting potency of 1.3 U/mg. In rabbit mammary explants, oPL stimulates casein synthesis. In a receptor assay for growth hormone using human liver, oPL and hGH are equipotent in competing for receptor sites, suggesting that oPL and hGH have common structural features that are lacking in

ABSTRACT. Ovine placental lactogen (oPL), has been purified approximately 1,000-fold from sheep cotyledons using conventional protein purification procedures. Radioreceptor assays using rabbit liver particulate fractions for growth hormone (RRA-GH) and using rabbit mammary gland particulate fractions for prolactin (RRA-PRL) were employed to monitor the hormonal activities. The molecular weight of oPL is approximately 22,000 as determined by gel filtration on Sephadex G-100, and its isoelectric point as determined by isoelectric focusing is 8.8. In the two RRA's, the displacement curve of oPL is parallel to

U

NTIL recently, human and monkey placental lactogen (hPL and mPL) were the only two placental lactogens that were fairly well defined. In earlier studies using bioassay procedures, placental lactogen had been identified in the rat (1) and in the goat (2). With the development of radioreceptor assays, research on other placental lactogens has been given a great impetus. The assay for detecting prolactin or lactogen (RRA-PRL) utilizes a rabbit mammary gland receptor (3), while the one for growth hormone-like activity employs a rabbit liver membrane receptor (4). By employing these two radioreceptor assays (RRA's), placental lactogens from several species have been detected and characterized. In our laboratory, we have identified and quantitated placental lactogen concentrations in the circulation of humans, monkeys, rats, mice, hamsters, guinea pigs, cows, goats, and sheep using these assays (5,6). In the present study, we report on the purification and characterization of ovine Received June 19, 1975. 1 Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0VV3. 2 Reproductive Physiology Section, Animal Research Institute, Ottawa, Ontario, Canada KIA 0C6. Contribution No. 587.

other non-primate hormones. (Endocrinology 98: 65, 1976)

placental lactogen from sheep cotyledons. Our studies indicate that there is a close homology between oPL and human growth hormone, thus confirming and extending an earlier report by Handwerger et al. (7-9). Materials and Methods Assays for monitoring hormonal activities Radioreceptor assays for prolactin or lactogen (RRA-PRL) and. for growth hormone (RRA-GH). Radioreceptor assays for measuring prolactin or

lactogen utilized a rabbit mammary gland receptor as described by Shiu etal. (3), while the one for measuring growth hormone, or growth hormonelike activity, employed rabbit liver receptors (4), with slight modification. A radioreceptor assay for hGH was also employed using human liver as a tissue source (10). Hormone preparations Human growth hormone (NIH-HS 1648E, 2U/mg), ovine prolactin (NIH-P-S-10, 26 IU/mg), bovine growth hormone (NIH-BG B1003A, 1.9 U/mg), and all other hormone preparations were kindly supplied by NIAMDD, NIH, USA. Protein measurement Protein concentrations of the fractions collected during purification were estimated by measuring the absorbance at 278 nm or by the method of Lowry et al. (11). 65

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66

CHAN, ROBERTSON AND FRIESEN

Concentration by ultra filtration All concentration procedures were carried out at 4 C in an Amicon Diaflo cell using UM-10 membranes. Purification procedures. All purification procedures were carried out at 4 C unless otherwise specified. Extraction. Placental tissues obtained at the time of surgery were immediately frozen at - 2 0 C without separating maternal and fetal cotyledons. For the extraction, 1.2 kg of placental tissue (5665 days' gestation) were homogenized with a Polytron PT-10 (Brinkmann) homogenizer at maximum speed for 30-60 seconds in 0.1M ammonium bicarbonate buffer adjusted to pH 9.5 with IN ammonium hydroxide, using a ratio of buffer to tissue of 5:1 (v/w). The homogenate was stirred overnight and then centrifuged at 30,000 x g for 30 min. The pellet was discarded. Ammonium sulfate precipitation. Ammonium sulfate was added slowly to the supernatant to achieve a 40% saturated solution. A precipitate was allowed to settle overnight, and then discarded. To the supernatant was added slowly additional ammonium sulfate to achieve a 75% saturated solution. After stirring the solution for 1 h and allowing the suspension to stand overnight, it was centrifuged to obtain the precipitate.

Endo • 1976 Vol 98 • No 1

ing oPL were collected, pooled, and concentrated to 50 ml by ultrafiltration. Gel filtration. The concentrate was applied to a Sephadex G-100 (Pharmacia, Uppsala, Sweden) column (4.2 x 104 cm) which was equilibrated with 0.01M ammonium acetate, pH 5.0. Ten ml fractions were collected in an LKB fraction collector. Carboxymethylcellulose (CMC) cation exchange chromatography. Appropriate fractions containing oPL from the Sephadex G-100 column (Fraction #56-80) were pooled and applied to a column (1.8 x 18 cm) of carboxymethylcellulose (Whatman CM-23) which was also equilibrated with 0.01M ammonium acetate buffer, pH 5.0. After washing the column with 300 ml of starting buffer, a stepwise elution with NaCl at 0.01, 0.05, 0.1,0.15, and 0.2M was carried out in the presence of 0.01M ammonium acetate, pH 5.0. The column was finally eluted with 0.5M NaCl. The fractions containing oPL (Fraction #950-1,150) were pooled and concentrated to a volume of 3 ml. Gel filtration. The concentrate was applied to a Sephadex G-100 column (1.4 x 94 cm) which was equilibrated with 0.1M ammonium bicarbonate, pH 8.7. The fractions (#33-40) containing oPL were pooled, concentrated to a volume of 1.5 ml, and lyophilized. Characterization of oPL

Dialysis. The precipitate was dissolved in 500 ml 0.1M ammonium bicarbonate. The solution was dialyzed initially against running tap water for 48 h, followed by dialysis against distilled water for a similar period. After dialysis, the solution was centrifuged at 100,000 x g for 90 min to obtain a clear supernatant. Diethylaminoethyl (DEAE) cellulose anion exchange chromatography. To the supernatant, solid ammonium bicarbonate was added to make a 0.05M solution, pH 7.8. The 0.05M supernatant

was applied to a column (6.5 x 40 cm) of DEAE cellulose (Whatman DE-32) previously equilibrated with 0.05M ammonium bicarbonate, pH 7.8. After the column was washed with 0.05M ammonium bicarbonate, pH 7.8, the concentration of ammonium bicarbonate in the buffer was increased stepwise to 0.1 and 0.2M. The column was finally eluted with 0.5M NaCl in the presence of 0.2M ammonium bicarbonate. Fractions contain-

Analytical gel electrophoresis. Polyacrylamide gel electrophoresis was carried out as described by Davis (12) and Reisfeld et al. (13) with slight modifications. Acrylamide gel electrophoresis, using 7.2% acrylamide and pH 8.8-9.0 was used, whereas in the Reisfeld procedure, 9.0% acrylamide and pH 4.3-4.5 buffer were employed. In both situations, one of a pair of identical gels was stained with a protein dye, while the other was divided at 1 mm intervals and each segment was eluted in 1 to 2 ml of 0.1M Tris-HCl, pH 7.6, containing 0.1% BSA at 4 C for 14 hours with shaking (Fisher Rotator). The eluants subsequently were analyzed by the two radioreceptor assays. All gels were stained in 1% amido black dye (in 7% acetic acid) for 1 or 2 h. The stained gel was removed and placed in a test tube with 7% acetic acid for destaining. Analytical gel isoelectric-focusing. Analytical thin-layer polyacrylamide gel isoelectric-


PURIFICATION OF OVINE PLACENTAL LACTOGEN focusing was performed according to the instructions supplied by the manufacturer of the LKB 2117 Multiphor apparatus. Separations were achieved using a pH range of 3.5 to 9.5 ampholine with a 6% (w/v) concentration of acylamide and a cross-linking of 2.5%. A maximum power of 4 watts and a focusing time of 4 h were used. Duplicate samples of the most highly purified oPL preparations were applied (75 /u-g each). One gel slice was stained with a 0.1% Coomassie brilliant blue R-250 containing 3.26% sulfosalicylic acid and 10.80% trichloroacetic acid (TCA), while the other was cut serially (0.5 cm) and the individual segments were eluted in 2 ml O.lM Tris-HCl, pH 7.6, containing 0.1% BSA (w/v) for 24 h at 4 C. Subsequently the eluants were analyzed by the two RRA's. The staining procedure was carried out at 22 C for 16 h, and then destained for 36-48 h in a solution containing water, ethanol, and acetic acid in a ratio of 8:3:1. One additional blank gel slice run at the same time was divided at 5 mm intervals and the consecutive individual segments were eluted with distilled water for 24 h at 4 C with shaking (Fisher Rotator). The pH of the eluants from each segment was determined (Fisher, Acumet, Model 420). Preparative isoelectric-focusing. The technique used was essentially that recommended in the 8100 Ampholine instruction manual (LKB). Separations were obtained using an isoelectric focusing column of 110 ml capacity (LKB) and carrier ampholytes in the pH range, 3.5-10, obtained from the same source. Gradients, routinely run with the cathode in the upper electrode position, were prepared in an LKB 8121 gradient mixer and loaded at 1-2 ml per min with a peristaltic pump (LKB). Electrofocusing with an initial power of 2.5 watts was allowed to proceed until a constant current was achieved (20-24 h). The gradient was displaced by the addition of water to the top of the column and 2 ml fractions were collected with the flow rate of 2 ml/min. Protein concentrations were monitored by absorbance at 278 nm, pH of the fractions was measured by the pH electrode (Fisher, Accumet Model 420), and the oPL concentration in the fractions was determined by the two RRA's. Gel filtration ofoPL. The elution volume of oPL was estimated on a Sephadex G-100 column (2.5 x 75 cm) which was equilibrated at room temperature with 0.01M Tris-HCl containing

67

0.1% BSA and 0.1M NaCl at pH 7.6, and was calibrated with [I25I]iodo hGH as marker protein. The hGH was labelled with 125I using the lactoperoxidase method described by Thorell and Johannson (14). Receptor assays. Radioreceptor assays for prolactin (RRA-PRL) and for growth hormone (RRAGH) using rabbit mammary gland and liver, respectively, and radioreceptor assays for human growth hormone using human liver were performed according to methods previously described (3,4,10). Except in the case of the radioreceptor assay for growth hormone using rabbit liver membrane receptors, [125I]iodo bGH and bGH were used as tracers and standard, respectively. [125I]iodo bGH was prepared by the lactoperoxidase method described by Thorell and Johannson (14) with slight modification. During iodination, the pH of 0.05M phosphate buffer added in the reaction mixture was pH 4.2, instead of pH 7.5, and 5 /xg of lactoperoxidase, 10 /JL\ of (1:1,500 dilution) of 30% hydrogen peroxide, and a reaction period of 20 min were used. The percentage of 125I incorporated into protein was 55-60, and the specific activity was 110-130 /xCi//xg protein. Biological. Bioassay of growth-promoting activity. Female Sprague-Dawley rats were obtained from Canadian Breeding Farm and Laboratory Ltd., Montreal, Canada. All rats were hypophysectomized at 2 weeks of age and at the end of a 4 week acclimatization period, animals manifesting inappropriate weight gains were discarded. The solutions containing saline, oPL or bGH were administered in 0.05 ml sc daily for a period of 9 days using 10 hypophysectomized rats at each dose. At the end of the experiment, all rats were killed and the sella turcica of each rat was inspected for completeness of hypophysectomy. The percentage body weight gain of individual rats was calculated and final results were expressed as the mean ± standard deviation for each treated group. The potency estimate of oPL using bGH (0.9 U/mg) as standard was calculated according to the method of Pugsley (15). Test for lactogenic activity. The lactogenic activity of oPL was assessed by its ability to stimulate casein synthesis in rabbit mammary explants maintained in organ culture. The procedures employed were similar to those reported by


68

Enclo • 1976 Vol 98 • No 1


Juergens et al. (16) for mouse mammary glands with slight modifications. Virgin New Zealand white rabbits (1-2 kg) were injected iv with 100 U hCG and 12 days later, mammary glands were removed under aseptic conditions, and cut into explants weighing approximately 1 mg. Four explants were placed on a siliconized lens paper, which was floated on 1 ml of culture medium in a Falcon culture dish. The culture medium was Medium 199 (GIBCO), which was supplemented with insulin and hydrocortisone, 10 /u,g/ml each, penicillin and streptomycin at concentrations of 50 /ug/ml, and HEPES (N-2-hydroxyethylpiperazine-N'^-ethanesulfonic acid) buffer (10 HIM). The dishes were placed in a plastic box (35 x 25 x 15 cm) and exposed to 95% air, 5% CO2 sufficient to maintain the pH at about 7.4, after which the system was closed and incubated at 37 C for 72 h. The explants were then transferred into new medium which contained either ovine prolactin (NIH-P-S-10) or a highly purified preparation of oPL at a final concentration of 1 /xg/ml. At the end of an additional 24 h incubation, [3H]leucine was added to the medium such that 1 ml ofmedium contained5£iCi of [3H]leucine, and the incubation was continued for an additional 4h. Casein assay. At the end of 4 h incubation, the explants from each dish were weighed and homogenized in 7 ml of solution with the following composition: KC1, 0.15 M; sodium phosphate (NaH2PO4), 0.004M; imidazole, 0.01M; Hammarsten bovine casein (Nutritional Biochemicals), 5 mg. The final pH was 6.7. The homogenate was centrifuged at 100,000 x g for 60 min, 5 ml of the supernatant fluid were made 0.01M with respect to CaCl2) and 50 fig of crystalline rennin (Sigma) was added, followed by incubation with shaking at 37 C for 30 min to precipitate the labelled and carrier casein. After centrifugation at 780 x g for 10 min at room temperature, the precipitate was washed twice with the homogenizing solution (without casein) made 0.01M with respect to CaCl2. The pellet was then heated in 5% TCA at 95 C for 15 min. After cooling, the suspension was centrifuged at 780 x g for 10 min at 4 C, washed twice with cold TCA, and three times with absolute alcohol-ether (3:1 v/v) at room temperature. The pellet was dissolved in 0.5 ml Protosol. After the addition of 0.1 ml distilled water, the dissolved pellet was counted in a toluene scintillation counter (Isocap/300, Nuclear Chicago). The results are expressed as cpm/mg wet weight of explants.

Results Purification Extraction of oPL from frozen placental cotyledons. Several buffers (pH 3.D-10.5) were examined to determine the optimal extraction of oPL from cotyledons that had been stored in the frozen state. The greatest amount of oPL was extracted at pH 9.5 (90 jxg RRA-PRL and 70 ixg RRA-GH were obtained per g wet weight). Acidic solutions were much less effective in solubilizing oPL. Moreover, attempts to recover oPL by re-extraction of the residue after acidic extraction using 0.1M ammonium bicarbonate, pH 9.5, were unsuccessful, and less than 1% of oPL could be recovered. Thus, it appears important that the primary extraction should be carried out at an alkaline pH at 4C. Diethylamino ethyl (DEAE) -cellulose anion exchange chromatography. Figure 1 shows the elution pattern when the dialyzed material after ammonium sulfate precipitation was subjected to DEAE ion exchange chromatography. Most of the oPL was unabsorbed by the column, whereas a considerable amount of protein was adsorbed. With increasing ammonium bicarbonate concentrations, little additional hormone was eluted; and in practice one need not elute with concentrations of ammonium bicarbonate greater than 0.05M to recover most of the oPL. Gel filtration. Figure 2 depicts the distribution of protein and oPL after gel filtration on Sephadex G-100 of the fractions containing oPL which were not adsorbed by the DEAE column (Fraction #50-300). Most of the oPL emerged in fractions with an elution volume 1.9-2.4 times that of the void volume. Carboxylmethyl cellulose (CMC) cation exchange chromatography. Fractions (#56-80) from the Sephadex G-100 column containing oPL were pooled and applied to a CMCcellulose cation exchange column. A step-


PURIFICATION OF OVINE PLACENTAL LACTOGEN

69

O.1M NH4HCC>3.O.2M NH4HCC>3O.2M NH4HCO3 pH82

p •O5MNaCI

8.0r

Absorbance RRA-PRL ' RRA-GH

FIG. 1. Diethylaminoethyl (DEAE)-cellulose chromatography of the oPL recovered from the ammonium sulfate precipitate. The DEAE-cellulose (Whatman DE-32) column (6.5 x 40 cm) was equilibrated with 0.05M ammonium bicarbonate,

pH 7.8.

3 0 0 4( FRACTION

NO.(15ml/tube)

wise gradient using NaCl in 0.01M ammonium acetate bluffer was applied. As shown in Fig. 3, most of the oPL was eluted with 0.2M NaCl, although a small additional peak eluted with 0.5M NaCl.

Characterization Analytical polyaerydamide gel electrophoresis. Figure 5 shows the protein pattern of the most highly purified oPL preparation upon electrophoresis at an operative pH of 8.9 and at pH 4.4. Ovine pituitary Gel filtration. The fractions containing oPL growth hormone and prolactin were also from CMC-chromatography (#950-1,159) examined at the same time for comparison at pH 8.9. In the most highly purified were pooled, concentrated and applied to a Sephadex G-100 column using O.lM am- preparation of oPL, three bands are still monium bicarbonate. The distribution of evident. When an unstained gel, in which oPL had been separated in a similar manner, oPL and proteins is shown in Fig. 4. A summary of the results obtained with the was divided and consecutive gel segments were eluted and assayed for oPL purification procedure appears in Table 1. -,60 7.0-

-140 O •—* Absorbance •—• RRA-PRL 0—0 RRA-GH

6.0-

FIG. 2. Gel filtration of the oPL-rich fractions obtained from the DEAE cellulose column shown in Fig. 2. In the Sephadex G-100 column (4.2 x 104 cm), 0.01M ammonium acetate, pH 5.0, was the eluting buffer.

20 1 100 b z n 80 5

iu5.0 u z < CD 4 . 0

2.0 1.0

0

VO

20

30

40

5 0 60 70 80 FRACTION NQOOml/Ujbe)



70

Endi) • 1976 Vol 98 • No !

001M NHJAcetate PH5

lOOh Absorbance RRA-PRL o—oRRA-GH

aso

UJ

o

i

Q60

ac o en

CD

(wo

200

300

400

500 600 700 800 TUBENQ(3ml/tube)

900

1000

1100

1200

FIG. 3. CM-cellulose chromatography of the oPL-rich fractions from the Sephadex G-100 column in Fig. 2 (Fractions #56-80). The CM-cellulose (Whatman CM-23) column (1.8 x 18 cm) was previously equilibrated with 0.01M ammonium acetate buffer, pH 5.0.

by the two RRA's, only the eluant from the segment corresponding to the middle band was active. Furthermore, when the most highly purified oPL preparation was sub-

jected to polyacrylamide gel electrophoresis at an acidic pH, three bands again were visible. Once more, when a duplicate unstained gel was divided and the segments

n560 0.65 -480 *—* Absorbance • — • RRA-PRL o—o RRA-GH

0.55

O 400

|

rn 320 O O

U z < Q35

z H240 m

CO

-160

>

Q15 80 0.05

2 4 616

20

24

28

32

36

40

44 ' 4'8 ' 52 ' 56

TUBE NO. (2ml/tube) FIG. 4. Gel filtration on Sephadex G-100 column (1.4 x 94 cm) of the fractions containing oPL from CM-cellulose column (Fractions #950-1,150). Sephadex G-100 column was equilibrated with 0.1M ammonium bicarbonate, pll 8.7.



71

TABLE 1. Table of purification

Procedure

Protein (g)

RRA-PRL (mg)

RRA-GH (mg)

Percentage recovery Protein

RRA-PRL

RRA-GH

Purification factor

Extraction, (0.1M NH4HCO3)

61.2

60.8

50.5

100

100

100

1

40-75% (NH 4 ) 2 S0 4 ppt.

10.6

42.4

33.9

16

70

67

4 29

DEAE-cellulose

0.6625

18.8

15.6

1

31

31

Sephadex G-100

18.2

14.2

0.41

30

28

CM-cellulose

0.255 *

10.8

8.4

—

18

16

—

Sephadex G-100

0.0061 f

6.0

5.1

0.001

10

19

1,000

70

* Not measurable—below the sensitivity of the procedure of Lowry et al. (11) (50 /Ltg/ml). f Dry weight. N.B. This procedure was employed for the purification of oPL using several different batches of placentas at different times, yielding a total of 25 mg oPL. In general, the results were similar to the data shown in this table.

eluted, only the eluant from the segment corresponding to the middle band contained oPL (Fig. 5). Analytical gel isoelectric focusing. In the highly purified preparation of oPL, a number of bands could be identified. When a duplicate gel slice that was not stained was divided into segments and eluted, oPL was found in eluants from segments that corre-

sponded to the two bands near the cathode (Fig. 6). In addition, when serial dilutions of the eluates from these segments were made, the response curves observed in the two RRA's showed parallelism with the ovine prolactin and hGH standards. Preparative isoelectric focusing. Figure 7 shows that when a partially purified oPL preparation obtained after CMC-chromatog-

oGH

ALKALINE oPRL

oPL

0.25

0.58

0.14

FIG. 5. Proteins were stained after polyacrylamide gel electrophoresis of oGH (76 /xg), oPRL (129 (jig), and oPL (74 Mg). The operative pH in all three alkaline gels was pH 8.9. The Rr values for these three hormones are 0.25, 0.58, and 0.14, respectively. The fourth gel depicts the stained bands seen after gel electrophoresis of oPL (75 fjLg) at an operative pH of 4.4. The dot beside the two gels to which oPL had been applied indicates the position where oPL was detected by two RRA's from eluants of another gel under similar conditions.

Rf



72

Endo • 1976 Vo! 98 • No 1

raphy was subjected to preparative isoelectric focusing, fractions with an absorbance at 278 nm were detected, but oPL was concentrated in fractions with pH 8.5-9.0.

10.0"

Gel filtration of oPL. Figure 8 shows that when the most highly purified oPL preparation (10 fxg) was applied to a Sephadex G-100 column together with [125I]iodo hGH, oPL virtually emerged in the same fractions. Since the molecular weight of [125I]iodo hGH under these conditions is estimated to be 20-22,000 daltons (17), it would seem reasonable to suggest that the molecular weight of oPL is similar. SLICE

FIG. 6. Distribution of oPL (bars) in eluants of gel segments after gel isoelectric focusing of an oPL preparation. The cathode was placed near segment 2, while the anode was placed on segment 22. OPL (75 fig) was applied on segments 4 and 22 (Whatman filter). Gel eluants were assayed, and the distribution of oPL was determined by the two RRA's. The protein bands from another longitudinal gel slice that was stained after isoelectric focusing of the oPL under the same conditions are shown below. The vertical black bands on the photograph of the gel represent the Whatman filter papers on which the sample was applied. The pH of the eluate fractions is indicated

Radioreceptor assays of the most highly purified oPL preparation. When the highly purified oPL preparation was assayed in both RRA's (Fig. 9a and b), oPL inhibited the binding ofeither [125I]iodooPRL (Fig. 9a), or [125I]iodo bGH (Fig. 9b) in a parallel manner to the hormone standards used (oPRL for RRA-PRL, and bGH for RRA-GH, respectively). When the ratio of prolactin to growth hormone activity of oPL was compared with that found for hGH in the two radioreceptor assays, it was apparent that oPL has a ratio of 2:1, whereas the ratio of hGH is 1:1. -.200

FIG. 7. The elution profile of CMC-purified oPL after preparative isoelectric focusing. Isoelectric focusing was performed in a sucrose gradient containing 0.7% carrier ampholytes in thepH range of 3.5 to 10.0.

10 15 20 25 30 FRACTION NO. (3ml/tube)

40


73


_ 2800 2400

FIG. 8. Gel filtration of oPL and [125I]iodo hGH. The Sephadex G-100 column (2.5 x 75 cm) was equilibrated at room temperature with 0.01M Tris-HCl containing 0.1% BSA and 0.1M NaCl at pH 7.6.

*—« 125I-hGH o-°RRA6H •-•RRAPRL

2000 1600 j= 1200 800 400

50 60 70 80 40 FRACTION NQ(2ml/tube)

When human liver membrane preparation was used for the radioreceptor assay (Fig. 10), the most highly purified oPL preparation was almost equipotent to hGH in inhibiting the binding of [125I]iodo hGH, whereas non-primate GH preparations were non-inhibitory even at very high concentra-

90

100

tions. Ovine prolactin also was without effect in the assay. In this assay hPL caused slight inhibition only at concentrations in excess of 1,000 ng/ml and hence, its cross reaction in this assay is less than 1%. These results suggest a close structural homology between oPL and hGH, because only hGH and oPL

hPL

X) XX) XXX) HORMONE CONCENTRATION, ng/ml

10 1OO HORMONE CONCENTRATION, ng/ml

FIG. 9. (a; left): Displacement curves for purified preparations of oPL, hGH, hPL, oPRL, and bGH in the radioreceptor assay for prolactin (RRA-PRL). Rabbit mammary gland particulate fractions were incubated with [125I]iodo oPRL in the presence of increasing concentrations of "cold" hormone. The ordinate represents the [125I]iodo oPRL bound to prolactin binding sites in the receptor preparation. In the absence of any added hormone the amount bound is taken to be 100%. (b; right): Displacement curves of purified preparation of oPL, hGH, hPL, oPRL, and bGH in the radioreceptor assay for growth hormone (RRA-GH). Rabbit liver membranes were incubated with [125I]iodo bGH in the presence of bGH, oPL, hGH, oPRL, and hPL. The amount of [l25I]iodo bGH bound in the presence of "cold" hormone is taken to be 100%.



74

Endo • 1976 Vol 98 • No 1

Test for lactogenic activity. Figure 12 shows the results of the assay for lactogenic activity of oPL. The most highly purified oPL is potent in stimulating casein synthesis using organ co-culture of rabbit mammary explants.

o 9

Discussion We have found that the placental lactogen content remains stable when placentas are stored at — 20 C for periods from 6 to 12 months, but after one year oPL content lqooo progressively decreases. Thus, frozen plaHORMONE (ng/ml) cental tissue stored for less than a year FIG. 10. Radioreceptor assay for human growth hormone provides a suitable tissue source for the using human liver. Human growth hormone (NIH-HS, purification of oPL. In our initial attempts to 1648 E, 2 U/mg) and [125I]iodo hGH were used as purify oPL from frozen placental cotyledons, standard and tracers, respectively. various difficulties were encountered. In the primary extraction procedure the alkaline cross react equally in all three receptor extract was turbid and viscous and could not assays. Moreover, of all pituitary and placen- readily be cleared by centrifugation or tal hormones tested, only oPL appears filtration. The viscosity of early-term placenequipotent to hGH in the radioreceptor tal extracts (54-65 days) was greater than assay using human liver. that of extracts obtained near term (130-145 days) or placental extracts obtained post Bioassay of growth-promoting activity. Fig- partum. The nature of the factors contributure 11 shows the results of the bioassay of ing to the viscosity is not clear, but muoPL for growth-promoting activity. The cous substances, such as mucin and mucorelative growth-promoting potency of oPL is polysaccharides, are suspected. When this calculated to be 1.3 U/mg, with 95% confi- thick extract was directly applied to a Sephadex G-100 column poor resolution was dence limits of 0.9-1.6 U/mg. hGH

30

SHEEP PLACENTAL LACTOGEN POTENCY- 1.3 U/mg 95%confidence limits (0.9-1.6 U/mg)

25

bGH !0.9 U/mg)

FIG. 11. Bioassay of growthpromoting activity of oPL.

' - - - -saline control-

.01

.02

.05

DAILY DOSE OF HORMONE INJECTED (mg)



75

active in the RRA but also caused growth in hypophysectomized rats. At this point it is unclear what factors contribute to the significant difference between the two oPL preparations. Only oPL and hGH react approximately 1400 equally in the two receptor assays, that is, have equal growth-promoting and lactogenic I vs i p-0.0002 activities. This observation hinted at the UJ I vs x p=0.004 possibility that oPL and hGH might also £1200 I v s i p = 0.4(n.s) u have certain common structural features, and suggested that it would be worthwhile examining the binding of oPL to a human tissue receptor for hGH. Lesniak et al. (18) 1000 have reported that human lymphocyte receptor for growth hormone is speciesspecific, and that non-primate growth hor•Imones fail to compete for the binding sites. 800 These same features were observed by Carr oPRL oPL none et al. (10) using a human liver radioreceptor assay. It was therefore of great interest to find FIG. 12. Test of lactogenic activity of oPL using organ co-culture of rabbit mammary explants. that oPL binds very effectively to human liver and inhibits the binding of [l25I]iodo obtained unless small batches of placental hGH, whereas hPL and non-primate growth cotyledons were used. The fractional pre- hormone preparations do not. These obsercipitation with ammonium sulfate (40-75% vations indicate that oPL has biological and saturation) proved to be a valuable step in perhaps structural and conformational features very similar to those of hGH. On the the purification of oPL. Although oPL is purified about 1,000-fold other hand, as Handwerger et al. (9) have by the method outlined, analysis of the most pointed out, oPL does not cross react highly purified preparation upon elec- immunologically with anti-hGH. In view of trophoresis revealed that it was not the similarity of oPL and hGH as judged by homogeneous. Eluants from the gels were the RRA's, it will be important to elucidate active in both radioreceptor assays, and there and the compare the sequences of oPL and was no evidence that the growth hormone- hGH. These studies might provide further like and prolactin-like activity resided in understanding of the sequence necessary for separate molecules. Indeed, the ratio of PRL binding to a human tissue receptor for to GH activity at all stages of the purification growth hormone. It is also conceivable that varied from 1:1 to 1:3. The oPL that we have oPL might be an effective growth-promoting purified differs somewhat from that reported agent in man, if the immunologic problems by Handwerger et al. (9). Their oPL prepara- are not prohibitive. Clearly, further studies tion had lactogenic activity very similar to along these lines are warranted. ours but significantly less somatotropic activAcknowledgments ity. They reported that oPL was only 1/5 as Special thanks are given to Dr. R. P. C. Shiu for active as hGH in the RRA-GH, whereas our performing the tests for lactogenic activity and to Dr. D. oPL preparation is almost equipotent with Carr for determining the binding to human liver hGH. Whereas Handwerger et al. (9) did not receptors. The helpful discussion and cooperation of perform a bioassay for growth-promoting Dr. P. A. Kelly, the technical assistance of Mrs. H. activity, our oPL preparation not only was Cosby, and the secretarial help of Miss H. Clark are 1600-


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gratefully acknowledged. We thank Mr. J. Harris for preparing the figures. This research was supported by the Medical Research Council of Canada and the National Institute for Child Health and Human Development (USPHS).

8. 9.

References 1. Astwood, E. B., and R. O. Greep, Proc Soc Exp Biol Med 38: 723, 1938. 2. Forsyth, I. A., In Wolstenholme, G. E. W., and J. Knight (eds.), Lactogenic Hormones, ChurchillLivingstone, London, 1972, p. 151. 3. Shiu, R. P. C , P. A. Kelly, and H. G. Friesen, Science 18: 968, 1971. 4. Tsushima, T., and H. G. Friesen,y Clin Endocrinol Metab 37: 334, 1973. 5. Kelly, P. A., H. A. Robertson, and H. G. Friesen, Nature 248: 435, 1974. 6. Kelly, P. A., R. C. P. Shiu, H. G. Friesen, and H. A. Robertson, Proc 55th Meeting Endocrine Soc, Chicago, Abstract No. 233A, 1973. 7. Fellows, R. E., T. Hurley, W. Maurer, and S.

10. 11. 12. 13. 14. 15. 16. 17. 18.

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Handwerger, Proc 56th Meeting Endocrine Soc, Atlanta, Abstract No. 116, 1974. Handwerger, S., W. Maurer, J. Barrett, T. Hurley, and R. E. Fellows, Proc 56th Meeting Endocrine Soc, Atlanta, Abstract No. 117, 1974. Handwerger, S., W. Maurer, J. Barrett, T. Hurley, and R. E. Fellows, Endocrine Res Commun 1: 403, 1974. Carr, D., and H. G. Friesen, J Clin Endocrinol Metab 1976 (In press). Lowry, O. H., N. J. Rosebrough, A. L. Farr, R. J. RandallJ Biol Chem 193: 165, 1955. Davis, B. J., Ann N Y Acad Sci 121: 404, 1964. Reisfeld, R. A., U. J. Lewis, and D. E. Williams Nature 195: 281, 1972. Thorell, J. I., and B. G. Johannson, Biochim Biophys Ada 251: 363, 1971. Pugsley, L. J., Endocrinology 39: 161, 1946. Juergens, N. G., F. E. Stockdale, Y. Topper, and J. J. Elias, Proc Natl Acad Sci USA 54: 629, 1965. Andrews, P., Biochem J 111: 799, 1969. Lesniak, M. A., J. Roth, P. Gorden, and J. R. Gavin, Nature [New Biol] 241: 20, 1973.


Purification and partial characterization of the cyanogen bromide fragments of ovine placental lactogen.

Isolation and structural characterization of ovine placental lactogen.

The purification and characterization of rat placental lactogen.

The purification and characterization of rabbit placental lactogen.

Immunofluorescence localization of ovine placental lactogen.

Stimulation of ovine placental lactogen secretion by arginine infusion.

Purification and characterization of mouse decidual calcyclin: a novel stimulator of mouse placental lactogen-II secretion.

Multiple forms of rat placental lactogen-II (rPL-II): purification and partial characterization of rPL-II.

Characterization of super-active insulin, prolactin and placental lactogen.

Purification and immunolocalization of ovine placental retinol-binding protein.

Synthesis and secretion of ovine placental lactogen and its biochemical properties.

Kinetics of placental lactogen in mid- and late-gestation ovine fetuses.

Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta.

Production of mouse placental lactogen-I and placental lactogen-II by the same giant cell.

Ovine placental lactogen: acute effects on intermediary metabolism in pregnant and non-pregnant sheep.

Distinct placental lactogen and prolactin (lactogen) receptors in bovine endometrium.

Effects of bovine growth hormone, human placental lactogen and ovine prolactin on intestinal fluid and ion transport in the rat.

Letter: Human placental lactogen measurements.

Human placental lactogen in pregnancy.

Letter: Human placental lactogen and fetal abnormality.

Letter: Human placental lactogen and diabetic pregnancy.

Letter: Human placental lactogen and fetal abnormality.

Purification of naturally occurring antibody to human placental lactogen by affinity chromatography.

Quantification and cellular localization of ovine placental lactogen messenger ribonucleic acid expression during mid- and late gestation.