Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones.

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.

Distribution of immunoreactivities for adenohypophysial hormones in the pituitary gland of the polypteriform fish, Polypterus endlicheri.

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunoreactive adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian alpha-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-beta-endorphin II as well as anti-mammalian alpha-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHbeta and salmon LHbeta, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.

Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.

A new preparative procedure without using ion-exchanger is described for the efficient purification of canine LH (cLH), FSH (cFSH) and TSH (cTSH) from the pituitary gland. The hormones were extracted from the pituitary homogenate with an ammonium sulfate solution, and were separated by Concanavalin (Con) A affinity-, hydrophobic interaction-, then immobilized metal ion affinity chromatography. In the immobilized metal ion affinity chromatography, we used copper (Cu2+) as chelated metal ion with ammonium ion gradient and pH gradient in phosphate buffer to attain separation of the hormones. High purity of cLH, cFSH and cTSH was indicated as single bands in SDS-PAGE, with apparent molecular masses of 34, 36 and 37 kDA, respectively. The purified hormones showed two bands corresponding to alpha (20 kDa) and beta subunits (cLH beta: 16 kDa, cFSH beta: 22 kDa, cTSH beta: 16 kDa) under reducing condition in SDS-PAGE. The purified hormones were prepared in good recovery (LH: 53%, FSH: 34%, TSH: 36%) with high biological activity or binding activity to the receptor. Cross-contamination of the purified hormone was less than 0.5%. Examination of the hormone fraction with isoelectric focusing showed that major peaks of isoelectric isoforms were maintained throughout the purification steps of cLH and cFSH, while a few peaks were lost in Con A affinity chromatography in cTSH purification. It was concluded that the present method could prepare highly purified cLH, cFSH and cTSH which retained isoforms of the hormones and biological activity or binding affinity to the receptor.

Renotropic activity in ovine luteinizing hormone isoform(s).

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

Purification and characterization of an incompletely glycosylated form of human chorionic gonadotropin from human placenta.

A small form of hCG (SP-hCG) was purified from an acetone powder preparation of human first trimester placenta by repeated gel filtration and ion exchange chromatography on a Q-Sepharose or FPLC Mono Q column. The estimated mol wt (Mr) of the small hCG by gel filtration is 43K compared to 58K for authentic hCG. The pI of SP-hCG is 10.0, suggesting deficiency of sialic acids. SP-hCG dissociates into subunits when treated with 6 M guanidine-HCl or analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two beta-subunits of SP-hCG were found with estimated Mr of 23K and 20K. Both are distinctly smaller than authentic hCG beta. A single alpha-subunit was found, with an estimated Mr of 21 K. The immunoactivity (8,900-10,000 IU/mg) of highly purified SP-hCG was comparable to that of reference hCG (CR119) determined by a RIA method using anti-hCG antibodies. The hCG/LH receptor-binding activity of SP-hCG is equivalent to that of reference hCG (CR119). Its biological activity is lower than that of reference hCG (approximately 30% or more) assayed by the in vitro stimulation of rat Leydig cells to produce testosterone and cAMP. A high dose is required to attain the same level of stimulation as reference hCG. The amino acid composition of SP-hCG is similar to that of reference hCG, whereas its hexsamine content is significantly lower. Its glucosamine content is about half that in reference hCG, while it completely lacks galactosamine. These findings suggest that SP-hCG is deficient in O-linked oligosaccharide chain in the beta-subunit, and that the N-linked oligosaccharide chains of both subunits are shortened. SP-hCG is one of the principal forms of the hormone present in first trimester placenta and may be a key intermediate in the posttranslational biosynthesis of hCG. Although it lacks O-linked sugar chains and shortened N-linked sugar chains, it possesses substantial biological activity. To have full biological activity, the hCG molecule must contain the complete complement of sugar chains.

Molecular forms of LH subunits in hypergonadotropic states.

To determine whether differences in subunits of LH exist in various hypergonadotropic states, we compared the elution profiles of immunoreactive alpha LH and beta LH following gel filtration over Sephadex G-100 (1.6 X 100 cm column) of sera and urinary acetone-insoluble precipitates from normal cycling women during the LH surge and untreated agonadal women and those receiving estrogen replacement. The elution profiles for the alpha subunit were similar in all subjects in both sera and urinary precipitates. In contrast, the elution profiles of beta LH varied between sera and urinary extracts among the 3 groups of subjects. Sera and urinary precipitates from the individual agonadal women eluted identically, but the profiles were distinct from those in samples from each of the cycling women. Exogenous estrogen therapy had no effect on the elution profiles of immunoreactive urinary beta LH in agonadal women but altered the ratios of beta LH/alpha LH excreted to those observed in normal cycling women. These data document the existence of immunoreactive subunits of LH in sera and urinary acetone-insoluble precipitates and confirm the existence of multiple forms of immunoreactive beta LH. The data further suggest that gonadal steroids may modulate the metabolism and/or excretion of beta LH in various hypergonadotropic states.

The production of active human thyroid-stimulating hormone from alpha and beta mRNAs in Xenopus laevis oocytes.

Active human thyroid-stimulating hormone (hTSH) was produced by Xenopus laevis oocytes following injection of an mRNA mixture of hTSH beta and alpha subunits synthesized by T3 RNA polymerase. Some of the hTSH molecules were secreted into the medium, while others remained in the cells. The active molecules consisted of alpha and beta subunits and were in highly glycosylated form. The Xenopus laevis oocyte-produced hTSH stimulated the rat thyroid cell line FRTL-5 to produce and secrete the cyclic AMP as does authentic hTSH.

[The characterization of free alpha subunit purified from the urine of normal term pregnant women].

Free alpha subunit (PU alpha) was extracted and purified from the urine of normal term pregnant women, and examined for molecular weight, electric charge, affinity to lectins (ConA, RCA and PNA) and the ability to combine with hCG beta in comparison with hCG alpha dissociated from hCG in vitro. The molecular weight of PU alpha was greater than that of hCG alpha in gel chromatography, but smaller in SDS-PAGE. However the free alpha subunits from the urine of patients with cancer were estimated to be larger than those of hCG alpha by both methods. In isoelectric focusing, while hCG alpha exhibited a neutral charge, PU alpha exhibited a negative charge. After treatment with sialidase, both hCG alpha and PU alpha were shifted to the basic region, indicating that they contained terminal sialic acid residues. The affinity to lectins indicated that PU alpha may contain both asparagine-linked and O-linked oligosaccharides, while hCG alpha contains asparagine-linked oligosaccharide only. PU alpha scarcely showed any combining activity with hCG beta, whereas hCG alpha combined actively. The O-linked oligosaccharide, which is not present in hCG alpha, may cause PU alpha to fail to combine with hCG beta.

Rat placental mRNA directs the synthesis of a polypeptide immunologically related to alpha-subunit of glycoprotein hormones.

In order to investigate the existence of a chorionic gonadotropin (CG) in the rat, placental mRNA was prepared from either the foetal disc or the maternal site of implantation in pregnant rats and translated in a wheat-germ cell-free translation system in the presence of 35S-labeled methionine and cysteine. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the radioactive material immunoprecipitated using antiserum specific to native rat alpha-subunit allowed us to isolate in translation products from both sources (foetal disc and maternal site of implantation) a single polypeptide of 17 kDa having the electrophoretic properties of the rat pituitary alpha-precursor. This polypeptide was absent in media derived from translation of mRNA extracted from uterine horn of non-pregnant female rats, and was approximately 10 times more abundant when RNA was derived from the maternal part (about 0.25% of cpm in total proteins) rather than the foetal part (0.026%) of the placenta. Immunoprecipitation was prevented in the presence of an excess of rat (but not ovine) alpha-subunit. Antisera directed against denatured bovine alpha-subunit, which cross-react with the rat pituitary alpha-precursor, did not bind the placental peptide. These results suggest that this rat placental polypeptide and the rat alpha-subunit of pituitary glycoprotein hormones have important differences in their primary structure, but share discrete structurally and/or conformationally related regions in their polypeptide chains. The possibility that these partial homologies account for gonadotropin-like activity of a presumed rat CG remains to be ascertained.

Studies of double-labeled mouse thyrotropin and free alpha-subunits to estimate relative fucose content.

The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free alpha-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free alpha-subunits. Thyrotropic tumor minces were incubated simultaneously with [35S]methionine and [3H]fucose. Thyrotropin and free alpha-subunits were labeled with both isotopes, and the ratio of 3H/35S was higher in free alpha-subunits than in thyrotropin; free alpha-subunits were approximately fivefold richer in fucose than was thyrotropin. The 3H/35S ratio was not substantially altered in TSH or free alpha-subunits secreted after a brief incubation with 10(-7) M thyrotropin-releasing hormone. Species which incorporated [3H]fucose were resistant to endoglycosidase H. Thus, mouse free alpha-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones.

Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography.

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

Sulfated LH subunits and a tyrosine-sulfated secretory protein (secretogranin II) in female rat adenohypophyses: changes with age and stimulation of release by LHRH.

Sulfated proteins are present in adenohypophyseal secretory granules but their function and structure are still largely unknown. We studied these proteins in homogenates from cow and rat adenohypophyses labeled in vitro with [35S]sulfate, by one-dimensional and two-dimensional polyacrylamide gel electrophoresis followed by fluorography. We found that the heterogeneous neutral-alkaline sulfated components of approximately 22-20 kDa and approximately 20-18 kDa previously described correspond to lutropin alpha and beta subunits sulfated on carbohydrates. During development sulfated lutropin subunits were found at highest levels in anterior pituitary glands of 14-day-old female rats. Secretogranin II, an acidic tyrosine-sulfated secretory protein, whose presence in granules of gonadotrophs has been recently described, had a similar distribution during development. In the 14-day-old female rat glands luteinizing hormone-releasing hormone stimulated the in vitro release of both sulfated lutropin subunits and secretogranin II. This finding further suggests that secretogranin II might be involved in the packaging of the gonadotrophin. Immature female rat adenohypophyses provide a useful approach for studying sulfation, both on carbohydrate and on tyrosine residues, of secretory proteins.

Characterization of glycoprotein hormone free alpha-subunit from human pituitary and placenta extracts.

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.

A gonadotropin-like thermostable peptide, prepared from bovine anterior pituitary, inducing spermiation in the frog Rana esculenta (L.) and binding to a rat ovarian FSH receptor.

A thermostable peptide, inducing sperm release in Rana esculenta L. and having immunological properties which resemble those of the gonadotropins but nonidentical with any one of the gonadotropins or their subunits, has been prepared from pars distalis of bovine anterior pituitaries by means of acetic acid extraction, heating, ethanol precipitation, and reverse-phase high-performance liquid chromatography (HPLC). During the purification, the biological activity was regularly followed by means of the potency of collected fractions to induce sperm release in frogs. Using HPLC (5 micron C18 silica), the activity was eluted with 38% of acetonitrile:water:trifluoroacetic acid (80:19.5:0.5) and 62% of 0.5% trifluoroacetic acid, pH 3.3. The active principle was also found to bind to a rat ovarian follitropin (FSH) receptor. In contrast, bovine FSH and LH treated identically to the peptide lost all such affinity, indicating that the peptide is not created from either of these during the purification procedure. Further, the peptide was, like FSH, also shown to stimulate aromatase activity in intact Sertoli cells from immature male rats in vitro.

Identification and partial characterization of a 25K protein structurally similar to prolactin.

Peptide mapping of individual pituitary proteins within the gel after separation by electrophoresis in NaDodSO4-polyacrylamide gels has revealed a high-molecular-weight (mol wt) protein whose fingerprint is similar to that of prolactin (PRL). This protein is approximately 4000 greater in mol wt than the traditional pituitary PRL, but does not appear to be the latter's prohormone. Its concentration ranged from 3 to 15% of the major PRL protein in the pituitary glands of several species examined. The protein isolated from sheep pituitary glands partly cross-reacted with a polyclonal antibody raised against the main PRL, but the material from mouse pituitary glands was completely noncross-reactive. The substance eluted from denaturing gels failed to significantly stimulate mucosal growth in the crop sac of the pigeons, a commonly used test for PRL's bioactivity. Its biological activities remain to be characterized.

Further characterization of the porcine LH subunits precursors in the translation products of pituitary RNAs.

We have shown that the precursors of porcine LH alpha and beta subunits have similar apparent molecular weights (Biochem. Biophys. Res. Comm., 1984, 118, 254). The aim of the present study was to characterize physiochemical features which permit to distinguish these precursors from each other. We report here that these precursors can be clearly distinguished by both their tryptic digests analyzed by one-dimension electrophoresis and their behavior in two-dimension electrophoresis whose first dimension was made in a non equilibrium pH gradient (2D-NEPHGE). The 2D-NEPHGE method developed in this study appears also useful to estimate simultaneously the translation activities of pituitary hormone mRNAs without a previous immunoprecipitation.

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells.

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of corticotropin-beta-lipotropin common precursor from ovine pituitary glands.

Ovine corticotropin-beta-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824-2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel electrophoresis, using immunoprecipitated and electrophoretically purified [125I]corticotropin-beta-lipotropin common precursor as a marker. The preparation was judged homogeneous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and beta-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-beta-lipotropin common precursor possessed specific activities of 116 micrograms of immunoreactive corticotropin and 210 micrograms of immunoreactive beta-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.