scholarly journals Studies on rat ovarian receptors for lutropin (luteinizing hormone). Interaction with β-subunit of sheep lutropin

1976 ◽  
Vol 160 (3) ◽  
pp. 615-619 ◽  
Author(s):  
K Muralidhar ◽  
N R Moudgal
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Chorionic Gonadotropin ◽  
Lower Order ◽  
Ovarian Response ◽  
Β Subunit ◽  
Α Subunit ◽  
Human Choriogonadotropin

By using radioimmunoassay, the interaction of sheep lutropin (luteinizing hormone, LH) β-subunit with rat ovarian receptors was investigated. The binding of β-subunit was specific, although of much lower order than that of lutropin. Sheep lutropin β-subunit effectively inhibited the binding of human choriogonadotropin (chorionic gonadotropin, gCG) to the ovary, showing that both occupy the same sites. The binding of sheep lutropin β-subunit to ovary was not followed by any detectable increase in cyclic AMP. The ovarian response to lutropin in terms of cyclic AMP production was inhibited in the presence of free β-subunit. The α-subunit of lutropin, when used at concentrations where contamination with whole lutropin was negligible, enhanced the degree of binding of β-subunit; this did not lead to increased cyclic AMP in the tissue. Surprisingly, the binding of β-subunit in vitro was drastically decreased by the prior removal of all endogenous rat lutropin bound to receptors. The implications of these data are discussed in the light of the reported biological activity of the β-subunit.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

hCG production and activity during pregnancy

2001 ◽  
Vol 12 (3) ◽  
pp. 191-208 ◽  
Author(s):  
H S Randeva ◽  
A Jackson ◽  
E Karteris ◽  
E W Hillhouse
Keyword(s):  
Luteinizing Hormone ◽  
Chorionic Gonadotropin ◽  
Essential Role ◽  
Follicle Stimulating Hormone ◽  
Thyroid Stimulating Hormone ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Hormone Specificity ◽  
Stimulating Hormone

Human chorionic gonadotropin (hCG) has an essential role in early pregnancy. It is a member of the glycoprotein hormone family also comprising the pituitary derived follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). Each hormone consists of a non-covalently bound α and β subunit. Within a species the α subunit is identical and hormone specificity is determined by the unique β subunit.

scholarly journals Studies on rat ovarian receptors for lutropin (luteinizing hormone). Factors influencing binding and response

1976 ◽  
Vol 160 (3) ◽  
pp. 607-613 ◽  
Author(s):  
K Muralidhar ◽  
N R Moudgal
Keyword(s):  
Luteinizing Hormone ◽  
Ovarian Response ◽  
Corpora Lutea ◽  
Human Choriogonadotropin ◽  
Factors Influencing ◽  
Pregnant Mare Serum Gonadotropin ◽  
Immature Rats ◽  
Lutropin Receptor ◽  
Serum Gonadotropin

The interaction of rat ovarian receptors with lutropin (luteinizing hormone, LH) in vitro was rapid and reversible. The degree of binding was saturable and susceptible to changes in the concentration of lutropin in the medium. The concentration of lutropin receptors in the ovary increases during the natural pubertal period and also in immature rats given pregnant-mare-serum gonadotropin and human choriogonadotropin. In the latter case, the increase in lutropin receptor, after injection of pregnant-mare-serum gonadotropin alone, could be detected only if the ovaries are freed of the bound gonadotropin before exposure to lutropin. The concentration of lutropin receptors was higher in the luteal compartment of the ovary than in the non-luteal parts and increased slightly in aged corpora lutea. Correlation between binding of lutropin to the ovary and the ovarian response to lutropin in terms of cyclic AMP production was found only in prepubertal rat ovaries and in young corpora lutea and not in aged corpora lutea, suggesting the non-equivalence of binding in vitro and ovarian response.

Heterogeneity of human lutropin. Detection and identification of α- and β-subunits

1985 ◽  
Vol 110 (2) ◽  
pp. 182-192 ◽  
Author(s):  
L. A. van Ginkel ◽  
J. G. Loeber
Keyword(s):  
Biological Activity ◽  
Isoelectric Focusing ◽  
Biologically Active ◽  
Ph Gradient ◽  
Β Subunit ◽  
Active Components ◽  
Α Subunit ◽  
Relative Response ◽  
Detection And Identification

Abstract. A highly purified LH preparation, prepared from human pituitaries (NM14) was studied using immunochemical and in vitro biological techniques. Using isoelectric focusing 5 different biologically active components could be detected, 4 of which were located between pH = 7.0 and 8.6, one was located at pH = 4.9. The biological activity in the acidic part of the pH gradient is probably due to the formation of an artefact during storage in solution. The experiments were performed with special emphasis on the occurrence of LH subunits, for which until now no pI-values have been reported. Using specific radioimmunochemical (RIA) systems at least 7 different α-subunit components and 4 different β-subunit components could be detected. The presence of even more components is likely. The α-subunit components, with pI-values of: 4.6, 5.2, 6.0, 7.1, 8.1, 8.8 and 9.7, were located spread over the entire pH-gradient whereas all β-subunit components were located above pH = 7.5, the pI-values being 7.7, 8.4, 8.5 and 10.4. The identification of these components as α- or β-subunit was based on the relative response in the different RIA systems, the absence of biological activity and the response changes during incubation at 37°C. Refocusing of the above mentioned biologically active components individually resulted each time in a single component with a pI-value identical to its corresponding 'parent'. After incubation at 37°C of these components each time the same variety of subunit components was found with discrete pI-values, identical to those above.

scholarly journals Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
So-Yun Lee ◽  
Munkhzaya Byambaragchaa ◽  
Seung-Hee Choi ◽  
Han-Ju Kang ◽  
Myung-Hwa Kang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Luteinizing Hormone ◽  
Chorionic Gonadotropin ◽  
Follicle Stimulating Hormone ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Α Subunit ◽  
Post Transfection ◽  
Glycosylation Sites ◽  
Equine Chorionic Gonadotropin

Abstract Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Results Both rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

PURIFICATION AND PROPERTIES OF THE SUBUNITS OF HUMAN PITUITARY FOLLICLE-STIMULATING HORMONE

1976 ◽  
Vol 69 (2) ◽  
pp. 263-273 ◽  
Author(s):  
RATNA C. SHOWNKEEN ◽  
ANNE STOCKELL HARTREE ◽  
FRANCESCA STEWART ◽  
K. MASHITER ◽  
V. C. STEVENS
Keyword(s):  
Biological Activity ◽  
Sialic Acid ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Β Subunit ◽  
Human Pituitary ◽  
High Biological Activity ◽  
Α Subunit

SUMMARY Highly purified human pituitary FSH was partially dissociated by treatment with 8 m-urea, and α- and β-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated α-subunit had less than 1%, and β-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with the purified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the β-subunit.

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