Journal Article Further Studies on the Purification of Human Pituitary Luteinizing Hormone Get access LEO E. REICHERT, JR., LEO E. REICHERT, JR. 1Department of Biochemistry and Department of Physiology, Division of Basic Health Sciences, Emory University Atlanta, Georgia 30322 Search for other works by this author on: Oxford Academic Google Scholar ALBERT F. PARLOW ALBERT F. PARLOW 1Department of Biochemistry and Department of Physiology, Division of Basic Health Sciences, Emory University Atlanta, Georgia 30322 Search for other works by this author on: Oxford Academic Google Scholar Endocrinology, Volume 75, Issue 5, 1 November 1964, Pages 815–817, https://doi.org/10.1210/endo-75-5-815 Published: 01 November 1964 Article history Received: 01 May 1964 Published: 01 November 1964
A detailed biological analysis and a simplified procedure for a method of serial extraction of acetone-preserved human pituitary glands is described. FSH is found almost exclusively in the first extract (at a mean potency of 2.1 XNIH-FSH-S1), together with a substantial amount of LH (0.13 XNIHLH- S1) and some TSH. A second extraction, under different conditions, yields GH, primarily in one fraction, at a potency approximately equivalent to the USP (bovine) Standard, together with prolactin. Also present in the second extract is LH (0.35 XNIH-LH-S1), and the greater part of the TSH. The third extraction yields additional GH, prolactin and ACTH. However, the recovery of ACTH is low. The method yields satisfactory amounts of GH, at a stage of purity suitable for use in the human, and recovers virtually all the FSH, LH and TSH, in forms readily susceptible to further purification. A modification and significant improvement of the bioassay procedure for GH, by body weight gain in immature hypophysectomized rats, is also described. (Endocrinology77: 1126,1965)
This chapter describes the induction of calcium transport into cultured rat Sertoli cells and liposomes by follicle-stimulating hormone (FSH). A substantial body of evidence indicates that precise control of cytosolic free Ca2+ is a critical requirement for Sertoli cell function. The involvement of FSH in regulation of Ca2+ flux into Sertoli cells suggests that cyosolic free Ca2+ may be an important second messenger in FSH signal transduction. The recently identified ability of FSH to bind calcium and to form transmembrane calcium-conducting channels, the presence of multiple mechanisms for regulating Ca2+ influx, that is, FSH-sensitive calcium channel activity and Na+/Ca2+ exchange attest to the importance of this ion in FSH action. Although the role of calcium in Sertoli cell function is not fully understood, initial data indicate that it has a profound effect on FSH-stimulated conversion of androstenedione to estradiol. It remains to be determined what other influences calcium may exert on Sertoli cell responsiveness to FSH stimulation.
We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a -60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 μg/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action. (Endocrinology126: 1318–1326,1990)
Luteinizing hormone preparations from a variety of sources were compared with respect to the slopes of their radioligand uptake inhibition curves. The system utilized human luteinizing hormone as the radioligand and homogenates of testis from mature rats as the receptor source. Based on the weighted mean values from replicate slope measurements, four major groups could be detected. In order of decreasing slope steepness, these were: Group I = human urinary LH, monkey pituitary LH, human pituitary LH and human chorionic gonadotropin; Group II = bovine pituitary LH, ovine pituitary LH, rat pituitary LH and equine pituitary LH; Group III = rabbit pituitary LH and chicken pituitary LH; Group IV = porcine pituitary LH and canine pituitary LH. There was no overlap of 95% fiducial limits for calculated slope values between groups. These groupings are at variance in some instances with those of a previous report (Endocrinology73: 509, 1963), based on slopes of dose response curves obtained using the ventral prostate assay for LH. These differences probably reflect the independence of the in vitro radioligand assay from influence by factors related to plasma half-life.(Endocrinology92: 646, 1973)
1. Purified preparations of human pituitary follicle stimulating hormone (FSH), prepared by different procedures, have been compared with each other and with human pituitary luteinizing hormone (LH) with respect to carbohydrate content and amino acid composition. 2. FSH is a glycoprotein containing about 78% protein. Its amino acid composition is not unusual. The carbohydrate component consists of hexose, hexosamine and sialic acid. 3. Compared to LH, FSH has a relatively high content of aspartic acid, glutamic acid, glycine, alanine, phenylalanine, lysine and histidine, and a lesser content of proline, valine and tyrosine. FSH also contains 3.7 times more sialic acid and 2.9 times more hexosamine than does LH. 4. On the basis of gel nitration and disc electrophoretic experiments, FSH appears to be a larger and more acidic protein than LH. (Endocrinology82: 109, 1968)
The role of follicle-stimulating hormone (FSH) in ovarian development was studied in immature rats. Autoradiographic analysis of gonadotropin binding sites revealed that FSH bound to granulosa cells while human chorionic gonadotropin (hCG) bound to thecal and interstitial cells in 25-day-old rats. Following 2 days of treatment with rat FSH (rFSH), hCG binding was observed in the granulosa cells of stimulated follicles. Histochemical localization of 3β-hydroxysteroid dehydrogenase activity also was apparent in granulosa cells from animals treated with FSH. Granulosa cells from rats treated with either hCG or diethylstilbestrol for 2 days failed to show consistent hCG binding. Isolated granulosa cells from intact and hypophysectomized rats treated with FSH demonstrated a greater binding capacity for hCG than granulosa cells removed from saline-treated animals. These results suggest that FSH administered in vivo may act on granulosa cells to induce or activate receptors for I_H (hCG) and 3β-hydroxysteroid dehydrogenase activity. (Endocrinology95: 818, 1974)
A method is described for electrophoretic purification of [125I]humah (h) FSH after radioiodination that improves radioligand binding to FSH membrane receptors. Lactoperoxidase-iodinated hFSH was separated from reaction products by electrophoresis on 7.5% polyacrylamide tube gels (PAGE). Material eluted from 3-mm gel slices was analyzed for incorporation of 125I and binding to antibody (RIA) or receptor (RRA), and by sodium dodecyl sulfate-PAGE for protein composition. Sodium dodecyl sulfate-PAGE analysis of individual PAGE fractions demonstrated that iodinated proteins, both higher and lower in apparent mol wt than intact FSH, were separated by PAGE, but not by gel filtration chromatography (Sephadex G-25). PAGE purification of radioligand resulted in significantly greater (compared to gel filtration) RRA sensitivity and specificity. Maximum binding of PAGE-purified [125I]hFSH to excess calf testes membrane receptors was 45%, with a specific activity of approximately 26 μCi/μg, as determined by the method of self-displacement. Maximum binding to excess hFSH antisera (NIH anti-hFSH 4) was 80–85%. This allowed a useful final dilution of 1:120,000, thereby facilitating development of a sensitive and specific RIA with this antiserum. These data indicate that PAGE separation of intact [125I]hFSH from other iodinated proteins results in improved radioligand binding, assay sensitivity, and assay specificity. In addition, PAGE-purified lactoperoxidase-iodinated hFSH is suitable for use in both RIA and RRA. (Endocrinology119: 1446–1453, 1986)
Porcine follicular fluid contains several factors capable of inhibiting the binding, in vitro, of follicle-stimulating hormone (FSH) to receptor, including an agonist and an antagonist of FSH biological activity in vitro. FSH receptor-binding inhibitory activity (FSH-BI) was determined with assays using radioligand (125iodide-human FSH) receptor (calf-testes membrane); in vitro biological assays (cultured immature rat Sertoli cells) were used to determine antagonist/agonist activity. FSH antagonist activity is due to a low (less than 5000) molecular weight FSH-BI that is soluble in acidic acetone and insoluble in diethyl ether allowing preparative scale isolation. Additional purification was achieved by anion-exchange and reverse-phase high-performance liquid chromatography. Highly purified, biologically active FSH-BI contained the amino acids Ser, Gly, Arg, Thr, Ala, Pro, Val, and Lys; hexoses (phenol-sulfuric acid-positive reaction); and ethanolamine. Thus, this FSH antagonist appears to be a complex glycopeptide--possibly derived from membrane components, as suggested by the presence of ethanolamine and carbohydrate residues. Porcine follicular fluid, therefore, contains a low molecular weight FSH antagonist that, along with the high molecular weight FSH agonist previously identified, may regulate gonadal responsiveness to FSH through interactions with the FSH receptor.
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