Expression and purification of human placenta lactogen in Escherichia coli.

There are many growth factors secreted by placenta including growth hormone, placenta lactogen (PL), prolactin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and chorionic gonadotropin. For a systematic study of how these growth factors work together to result in the various biological functions and future clinical applications, it is needed to produce enough quantities of each protein. In this paper, we report the cloning of human PL (hPL) and expression by Escherichia coli (E. coli). Four kinds of expression vectors containing the hPL gene were transformed into several kinds of suitable host strains and grown at 37 and/or 30 degrees C. Determination of the yield of recombinant hPL by SDS-PAGE reveals that among the various conditions, pQE30-PL in E. coli strain M15[pREP4] expressed the largest amount of recombinant hPL at 37 degrees C. However, the expressed recombinant hPL was accumulated in inclusion body forms. The inclusion bodies were solubilized in 8M urea and purified by a His6 tagged affinity column under denaturing condition and the final yield of hPL was determined to be 48 mg/L. Intra-chain disulfide bonds could be formed either by oxidation in the refolding buffer or by air oxidation in the presence of urea. The biological activity was examined by the fact that hPL could stimulate erythroid maturation by the formation of hemoglobin in K-562 cells in the presence of erythropoietin. Initial optimization studies resulted in the production of 282.4 mg/L of hPL.

Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen.

Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta. The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector. E. coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa. Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL. Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected. The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.

Crystallization of ovine placental lactogen in a 1:2 complex with the extracellular domain of the rat prolactin receptor.

Growth hormone and prolactin control somato-lactogenic biology. While high-resolution crystal structures have been determined for receptor complexes of human growth hormone, no such information exists for prolactin. A stable 1:2 complex was formed between ovine placental lactogen, a close prolactin homologue, and two copies of the extracellular portion of the rat prolactin receptor. Using synchrotron radiation, native data have been collected to 2.3 A. Crystals contain one complex per asymmetric unit. The crystal structure of this complex will shed light on the structural reasons for cross-reactivity and specificity among the endocrine hormones, placental lactogen, prolactin and growth hormone.

Rat placental lactogen-I variant (rPL-Iv), product of an unique gene, is biologically different from rPL-I.

The rat placenta expresses two rat placental lactogen-I (rPL-I) proteins: the normal rPL-I in the first half of pregnancy and a variant (rPL-Iv) in the second half of pregnancy. They are 70% identical at the amino acid level but arise from different cell types: rPL-I from giant cells and rPL-Iv from cytotrophoblasts. To assess whether rPL-Iv originates from alternative splicing of the rPL-I gene or is the product of a separate gene, genomic clones of rPL-I and rPL-Iv were isolated from a lambda EMBL-3 rat genomic library. Restriction enzyme analysis of the 14-kilobase full-length genomic clones of rPL-I and rPL-Iv indicated that the two genes are distinct. To assess the biological activity of the variant protein relative to other members of the rat PL/PRL/GH family, two expression systems were chosen to obtain the purified recombinant protein: 1) a secreted form of rPL-Iv was obtained from Chinese hamster ovary (CHO) cells transfected with rPL-Iv-complementary DNA; and 2) a rPL-Iv fusion protein (Bac-rPL-Iv) was obtained from Spodoptera frugiperda (Sf9) insect cells that had been infected with a recombinant baculovirus generated from rPL-Iv complementary DNA. An antibody was generated to the purified Bac-rPL-Iv fusion protein and used for affinity chromatography to purify recombinant rPL-Iv from the CHO cell media. The mitogenic activity of rPL-Iv was monitored in the Nb2 lymphoma cell bioassay. The relative potency of rPL-Iv compared with other members of the PL/PRL/GH family follows: ovine PRL 100, rPL-I 200, rPL-II 160, rPRL 40, CHO-rPL-Iv 0.7, and Bac rPL-Iv 0.4. Iodinated CHO-rPL-Iv showed minimal binding to Nb2 lymphoma cells, but at a 500-fold protein concentration rPL-I was able to displace [125I]rPL-I from the lymphoma cell PRL receptor. Using recombinant CHO-derived rPL-Iv as standard and antisera against the Bac-rPL-Iv fusion protein, a RIA was developed for rPL-Iv. In pregnant rats rPL-Iv appeared in the serum at day 14, rising to a peak of 2080 +/- 440 ng/ml at day 18, followed by a slight decline. These values reflect the levels of messenger RNA for rPL-Iv in rat placenta noted previously. In summary, rPL-Iv arises from a gene different from rPL-I and the rPL-I protein displays minimal binding and mitogenic activity in the Nb2 lymphoma cells.

Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone.

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.

Placental lactogen-like proteins in the rabbit placenta.

Rabbit placentae and embryos at days 11 and 12 were analyzed by two-dimensional gel electrophoresis and by light microscopic histology for the presence of placental lactogen-like proteins. Immunoblotting and immunohistochemistry were performed by using a goat anti-human placental lactogen serum as well as a monoclonal mouse anti-human prolactin immunoglobulin; the results were similar. In the gel electrophoresis of placental tissue, three protein spots at pH 5.6 and 43, 39, and 35 kDa were immunostained; they were absent in the embryo. Immunoresponse was restricted to the cytotrophoblast. Immunofluorescent cells were mainly found on the proximal parts of the placental trabeculae.

Novel in-frame two codon translational hop during synthesis of bovine placental lactogen in a recombinant strain of Escherichia coli.

A recombinant Escherichia coli strain was constructed for the overexpression of bovine placental lactogen (bPL), using a bPL structural gene containing 9 of the rare arginine codons AGA and AGG. When high level bPL synthesis was induced in this strain, cell growth was inhibited and bPL accumulated to less than 10% of total cell protein. In addition, about 2% of the recombinant bPL produced from this strain exhibited an altered trypsin digestion pattern. Amino acid residues 74 through 109 normally produce 2 tryptic peptides, but the altered form of bPL lacked these two peptides and instead had a new peptide which was missing arginine residue 86 and one of the two flanking leucine residues. The codon for arginine residue 86 was AGG and the codons for the flanking leucine residues 85 and 87 were TTG. When 5 of the 9 AGA and AGG codons in the bPL structural gene were changed to more preferred arginine codons, cell growth was not inhibited and bPL accumulated to about 30% of total cell protein. When bPL was purified from this modified strain, which included changing the arginine codon at position 86 from AGG to CGT, none of the altered form of bPL was produced. These observations are consistent with a model in which translational pausing occurs at the arginine residue 86 AGG codon because the corresponding arginyl-tRNA species is reduced by the high level of bPL synthesis, and a translational hop occurs from the leucine residue 85 TTG codon to the leucine residue 87 TTG codon. This observation represents the first report of an error in protein synthesis due to an in-frame translational hop within an open reading frame.

Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen.

Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation. However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity. The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity. Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells). Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure. Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL. Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties. It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors. Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.

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

The present study was designed to develop a procedure for purifying rPL-II and a homologous radioimmunoassay (RIA) for rPL-II. Molecular profiles of rPL-II were also investigated in tissue and plasma. rPL-II was purified 3,780-fold, based on its radioreceptor assay (RRA) activity compared to ovine prolactin (0PRL), from the placenta of day 18 pregnant rats using ammonium sulfate precipitation and chromatography on phenyl-Sepharose, DEAE-TOYOPEARL 650S, AF-chelate TOYOPEARL 650M and Sephadex G-100. Electrophoretic analysis on SDS gel revealed molecular weight heterogeneity of purified rPL-II, which consisted of three proteins; a major form with a molecular weight of 20.0 K and two minor forms with molecular weights of 20.6 K and 21.0 K under non-reducing conditions. One of the minor forms of rPL-II observed under non-reducing conditions disappeared with 2-mercaptoethanol treatment and the rest of the hormones migrated as 24.5 K and 25.0 K molecular weight species, suggesting that it is a cleaved form of rPL-II. Purified rPL-II displaced 125I-labelled oPRL from binding sites on rabbit mammary gland membranes in a dose-dependent manner. rPL-II and rPRL were, respectively, 21 and 2 per cent as effective as oPRL in the displacement. Antibody to purified rPL-II was raised in rabbits and a homologous RIA for rPL-II was developed. No displacement was observed with rPRL, rGH, oPRL, and other pituitary hormones up to 1,000 ng/ml. Molecular profiles of rPL-II in the placental tissue and plasma from day 18 pregnant rats were examined by gel chromatography on Sepharcryl S-300 HR and by Western blotting. Chromatography of the placental extracts revealed a single peak, which accounted for 86 per cent of the total RIA activity. Anti-rPL-II antiserum detected proteins of at least three molecular sizes as monomeric forms with molecular weights of 20.0, 20.6, and 21.0 K in the non-reducing placental extracts. One of them disappeared with 2-mercaptoethanol treatment and other two proteins had molecular weights of 24.5 and 25.0 K, indicating monomeric heterogeneity of rPL-II in the tissue. The elution profile of day 18 plasma in RIA activity gave two major peaks; the first, eluting just after the void volume (approximate molecular weight of 530 K) accounted for 35 per cent of the total RIA activity, and the second coinciding with the same elution volume as the monomeric form in the placental extract constituted about 26 per cent of the total RIA activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Two distinct placental lactogen-like substances in serum during mid-pregnancy in the rat.

Two forms of placental lactogen (PL), designated PL-I and PL-II, have been reported in the serum of pregnant rats; PL-I was secreted during mid-pregnancy and PL-II was secreted near term. In the present study, we found that two distinct forms with PL-like activity were secreted into the blood during mid-pregnancy. Serum from day-12 pregnant rats was subjected to high-performance liquid chromatography (HPLC) gel-filtration (TSK-G3000SswXL column), and PL activity was assayed by radioreceptor assay using 125I-labeled rat PRL and hepatocyte plasma membrane from pregnant rats. HPLC-gel filtration separated the PL activity into two peaks with mol wt of 55-60 K and 45-50 K, as estimated by reference to the elution volume of standard proteins. We tentatively designated these peaks PL-alpha and PL-beta, respectively. Considering these mol wt, PL-beta seemed to be identical to PL-I. mRNA was extracted from samples of placenta obtained each day from day 8 to day 12 of pregnancy and analyzed by means of a translation system involving micro-injection into Xenopus oocytes. The time of appearance of the mRNAs corresponding to PL-alpha and PL-beta did not correspond and differed according to the day of pregnancy, suggesting that there are individual mRNAs for each PL in rat placenta. Treatment of PL-alpha and PL-beta with peptide: N-glycosidase F completely abolished the binding activity to their receptors. Also since they were sensitive to glycosidases such as endoglycosidase H, endoglycosidase F and neuraminidase, these PLs were considered to possess N-linked glycoresidue(s) and to be sialylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Placental lactogen secretion during prolonged-pregnancy in the rat: the ovary plays a pivotal role in the control of placental function.

The serum of rats at mid-pregnancy contains at least 2 distinct placental lactogen (PL)-like substances tentatively termed placental lactogen-alpha (PL-alpha) and placental lactogen-beta (PL-beta) (Endocrinol Japon 38: 533-540, 1991). We have investigated the secretory patterns of three placental lactogens (PL-alpha, PL-beta and placental lactogen-II) during normal pregnancy and in two prolonged-pregnancy models. Pregnancy was prolonged by the introduction of new corpora lutea by inducing ovulation on day 15 of pregnancy by successive treatments with PMSG (30 IU/rat, sc on day 12) and hCG (10 IU/rat, iv on day 14), and in the second model by progesterone implants on day 15 of pregnancy. During normal pregnancy, each of the 3 PLs exhibited only one secretory peak in the serum; PL-alpha and PL-beta on day 12 and placental lactogen II (PL-II) on day 20. Interestingly, in the rats with new sets of corpora lutea, serum PL-alpha and PL-beta levels began to increase again on day 18 and showed peaks on day 20 for PL-alpha and on day 22 for PL-beta. In this model, the initiation of PL-II secretion was not affected, but high levels were maintained until day 26, when parturition occurred. In rats receiving either PMSG or hCG, the secretory patterns of the PLs were similar to as those during normal pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, partial characterization, and development of a specific radioimmunoassay for goat placental lactogen.

Placental lactogen (PL) was isolated from goat cotyledonary tissue by a combination of mild alkaline extraction, anion and cation exchange chromatography, chromatofocussing and molecular filtration. The product, enriched 15,000-fold from the initial extract, was homogeneous when examined by SDS-gel electrophoresis (Mr 22,500) and isoelectricfocussing indicated a pI of 8.35 with a trace contaminant of pI 8.0. When assessed by relative binding activity in radioreceptor assays (RRA), goat PL exhibited somatotropic activity equivalent to 2.2 units/mg dry weight and lactogenic activity equivalent to 28.5 units/mg. A radioimmunoassay (RIA) for goat PL is described that is highly sensitive (190 pg/tube) and has acceptable repeatability within and between assays (6 and 13%, respectively). The assay is not affected by goat pituitary extracts or partly purified goat growth hormone and prolactin. Despite the marked increase in sensitivity of the RIA over that previously available when goat PL was measured by RRA, the hormone was not detected in jugular plasma of goats before Day 44 of pregnancy; concentrations increased thereafter and highest levels were measured during the last third of pregnancy in animals bearing triplets. Measurements by RIA are in general agreement with those obtained earlier in several studies in which RRAs were used. The hormone was detected in amniotic fluid. Maternal concentrations of goat PL declined before parturition and were undetectable by 18 h post partum.

Purification and structural characterization of ovine placental lactogen.

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4.2 mg of oPL, with an approximately 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0.18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4.1 nmol/l) and oPRL (ED50 = 1.1 mumol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a lambda ZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides).(ABSTRACT TRUNCATED AT 250 WORDS)

[The radioimmunological determination of human placental lactogen with the RIO-PL-125I reagent kit]. / Radioimmunologicheskoe opredelenie platsentarnogo laktogena cheloveka naborom reaktivov RIO-PL-125I.

The authors described the development of a radioimmunoassay for the determination of human placental lactogen (PL) in the blood serum of pregnant women. Methods of obtaining RIA components were developed: highly refined and stable agents of the labeled and unlabeled hormones, specific antisera and a highly effective separating agent. The test system and commercial kits were used for comparative determination of the blood PL concentration at various terms of pregnancy. Similar results were obtained. The above test system was laid in the basis of a commercial kit RIO-PL-125I (USSR).

[Placental growth hormone. Significance relative to growth hormone and lactogen hormone]. / Hormone de croissance placentaire. Signification par rapport aux hormones de croissance et lactogène.

Normal human placenta secretes within maternal compartment a pregnancy associated protein, placental growth hormone (PGH). This entity, agonist of pituitary GH, appears responsible for the elevated IGF I blood levels observed in the mother during pregnancy, while pituitary GH is no longer secreted. PGH could thus play a significant role in the anabolic processes of pregnancy. The biochemical mechanism responsible for PGH production is the expression of the GH-V gene at the placental level. This has been demonstrated by the positive probing of GH-V mRNA in this tissue, by the establishment of the restriction map and sequence of its cDNA as well as by the NH2-terminal sequence determination of both 22 and 25K PGH forms. The positive aspect of this function for human reproduction gains weight in that a placenta deleted for the CS-A, GH-V and CS-B gene expresses alternative genes coding for protein similar or related to pituitary GH or hPL, respectively.

Ovine chorionic somatomammotropin and foetal growth.

Ovine chorionic somatomammotropin (oCS) enhances the weight and bone growth of hypophysectomized rats. It acts as a bifunctional hormone, since it binds both to lactogenic and somatotropic receptors. Ovine foetus weight gain is closely related to oCS and oGH serum levels. oCS is able to stimulate somatomedins by foetal liver. Moreover, oCS specific receptors are present in some foetal tissues. So, all these facts involve oCS in foetal growth, whereas pituitary growth hormone intervenes in postnatal growth. A study of structure function relationships between growth hormones and placental hormones is exposed in order to localize somatotropic sites.

Purification and partial characterization of two prolactin-like glycoprotein hormone complexes from the midpregnant mouse conceptus.

Two PRL-like glycoprotein hormone complexes were purified from the medium of cultured mouse conceptuses from day 10 of pregnancy: mouse placental lactogen-I (mPL-I) (29-32K), and mPL-I (36.5-42K). Sodium dodecyl sulfate-gel electrophoresis revealed that mPL-I (36.5-42K) is a complex of five proteins with mol wt of 36.5K, 37.5K, 39K, 40.5K, and 42K. Deglycosylation with peptide: N-glycosidase F or trifluoromethanesulfonic acid produced a single 29K protein. mPL-I (36.5-42K) was also sensitive to neuraminidase, but not to endo-beta-N-acetylglucosaminidase H or bacterial alkaline phosphatase. The production of intermediates from partial digestion of mPL-I (36.5-42K) with endo-beta-N-acetylglucosaminidase F indicated the presence of multiple glycosylation sites. mPL-I (29-32K) is a complex of three proteins with mol wt of 29K, 30.5K, and 32K. Treatment with peptide:N-glycosidase F or trifluoromethanesulfonic acid reduced the mol wt of the 30.5K and 32K bands to 28K. The 30.5K band was sensitive to endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, but the 32K band was not. Neither band was sensitive to neuraminidase or bacterial alkaline phosphatase. The 29K band was resistant to all chemical and enzymatic treatments and is probably not glycosylated or phosphorylated. In the nonreduced state, neither form of mPL-I showed an increase in mobility over that of its reduced counterpart on sodium dodecyl sulfate-gel electrophoresis, indicating that neither form of mPL-I contains the large disulfide loop common to hormones of the PRL family. After iodination, all component proteins of both forms of mPL-I were found to bind to day 17 pregnant mouse liver membranes and were displaceable by excess mPL-II. In a radioreceptor assay, 125I-labeled mPL-I (36.5-42K) was displaced by mPRL or mPL-II, but not by mGH. An antiserum to both forms of mPL-I was generated, and a RIA employing mPL-I (36.5-42K) as the standard and radioligand was developed. Dilutions of day 10 pregnant maternal mouse serum and placental homogenate and a partially purified fraction of mPL-I (29-32K) produced displacement curves parallel to that of mPL-I (36.5-42K) standard curve. Five micrograms of mPRL, mPL-II, or mGH or 10 microliter day 17 pregnant or male mouse serum did not displace the radioligand from the antibody. mPL-I (36.5-42K) was lactogenic, but it did not possess LH-like bioactivity.

Isolation and characterization of multiple forms of bovine placental lactogen from secretory granules of the fetal cotyledon.

Previous work has shown that, unlike other species, placental lactogen (PL) in the bovine (bPL) has a mol wt of approximately 32,000 and exists in several different forms with different isoelectric points. This study was carried out to develop a more rapid purification scheme, whereby the yield of bPL obtained was increased while at the same time the possibility of artifacts from a prolonged purification protocol was decreased. A procedure was developed in which a fraction enriched in bPL-containing granules was obtained after gentle disruption of the binucleate cells of the fetal cotyledon. The fetal portion of the placentomes from midpregnant cows was minced with scissors and vigorously stirred in order to remove and disrupt binucleate cells within the fetal villi. The supernatant from this step was fractionated by differential centrifugation followed by a four-step discontinuous Percoll gradient of 1.03-1.08 g/ml. A granule-enriched fraction was isolated from the 1.04 g/ml zone from which membrane-enclosed protein was released by freezing and thawing. Membranes and insoluble proteins were sedimented by high speed centrifugation to yield an extract which contained approximately 20% of the hormone initially in the tissue. Two subsequent chromatographic steps, gel filtration on Sephadex G-75 and high performance reversed phase chromatography with a C-4 column, resulted in a preparation of greater than 98% homogeneity. Two-dimensional gel electrophoresis of purified bPL revealed at least nine protein spots in the 31,000-33,000 mol wt range with isoelectric points ranging from 4.85-6.3. All forms exhibited parallel dilution curves in a RIA for bPL. It would appear, therefore, that multiple forms of bPL exist and that they are not artifacts of the prolonged purification protocol previously used.

Purification and partial characterization of hamster placental lactogen.

A radioreceptor assay for PRL-like activity was used to monitor the purification of a lactogenic protein, hamster placental lactogen (haPL), from late pregnant hamster placentae. Investigations of the PRL-like activity in placental extracts demonstrated that haPL is subject to disulfide-related aggregation phenomena that are not observed for mouse PL. By inclusion of 2-mercaptoethanol in the buffers used for purification, monomeric haPL was obtained. A 750-fold purification was achieved by ammonium sulfate precipitation and chromatography on phenyl-Sepharose, TSK diethylaminoethyl-650S, hydroxylapatite, and Sephadex G-100. This procedure yielded 6.2 mg (by dry wt) of purified haPL from 286 g 15-day pregnant hamster placentae, with an overall yield of 20%. The purified haPL has a mol wt of 25,200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 8.8. haPL is lactogenic as judged by its ability to compete for lactogen binding sites on rabbit mammary gland membranes and to stimulate secretion of alpha-lactalbumin by cultured mouse mammary gland epithelial cells.

Purification of ovine placental lactogen (oPL) using high-performance liquid chromatography. Evidence for two forms of oPL.

After initial purification of ovine placental lactogen (oPL) using the procedures described previously [(1976) Endocrinology 98, 65-75], the oPL preparation was further purified by high-performance liquid chromatography (HPLC) using an anionic exchange column (Bio-Sil TSK DEAE-2-SW). Two forms of oPL with different relative mobilities on HPLC were isolated and designated oPL-I and oPL-II. Subsequent analysis by polyacrylamide gel electrophoresis containing SDS revealed that oPL-I and oPL-II are nearly homogeneous (greater than 90% pure) and are identical in apparent Mr (approx. 22 000-23 000). Like human growth hormone (hGH), oPL-I and oPL-II are equally active in the radioreceptor assays for growth hormone-like activity (RRA-GH) and for prolactin-like activity (RRA-RRL). In the radioimmunoassay of oPL, both oPL-I and oPL-II are immunologically similar. Analysis of amino acid composition revealed that oPL-I and oPL-II consist of 199 and 196 residues, respectively, and have almost identical residues except that oPL-I has a higher content of glycine. Furthermore, both oPLs have a general similarity in amino acid composition to oGH and oPRL except for a lower content of methionine and leucine but with a higher content of lysine. Our studies demonstrated the presence of two similar forms of oPL. Whether these two similar forms of oPL share identical primary structure remains to be determined.