A comprehensive series of overlapping synthetic peptides have been used to study the relationship between the primary structure of the ovarian receptor for LH/human CG (hCG) and hormone binding. Twenty-four consecutive, overlap peptides that replicate the entire extracellular domain of the rat luteal receptor have been synthesized by standard solid-phase techniques on an automated synthesizer. Eight additional peptides from the extracellular domain and three peptides replicating the putative extracellular loop regions have also been synthesized. Each peptide was evaluated in RRAs for interaction with hCG by measuring its ability to competitively inhibit binding of 125I-hCG to membrane receptor. Twelve peptides were found to be potent in RRAs and caused a reduction of half-maximal binding of 125I-hCG at concentrations of 10-250 x 10(-6) M. The 12 active peptides (and adjacent inactive peptides) defined at least 4 independent receptor regions that can interact with hormone. One site near the NH2-terminus was localized to receptor residues Arg21-Pro38. Two more sites of hormone interaction were identified by peptides replicating residues Arg102-Thr115 and Tyr253-Phe272. A fourth binding region was identified in the third putative extracellular loop, replicated by rat luteal receptor peptide Lys573-Lys583. The amino acid sequences of the four active rat LH/hCG receptor regions were aligned and compared with published sequences for other glycoprotein hormone receptors. Three regions (Arg102-Thr115, Tyr253-Phe272, and Lys573-Lys583) showed high sequence homology with the human LH/hCG receptor, human TSH receptor, and rat FSH receptor and may represent contact sites for the alpha-subunit of hormone. The other binding region, Arg21-Pro38 had low sequence homology with the other glycoprotein hormone receptors and is postulated to be a binding determinant for beta-hCG/LH. This report demonstrates that synthetic overlap peptides of confirmed sequence can be used to successively identify hormone interaction sites of glycoprotein hormone receptors.

Twenty-seven synthetic peptides, representing the entire structure of the human glycoprotein hormone alpha-subunit were used to map the antigenic structure of the alpha-subunit. Solution phase and solid phase assays were performed with these peptides and a panel of eight monoclonal antibodies (MAb). Two dominant regions were localized between residues 22-37 and 70-87. All eight antibodies recognized these regions, but differed somewhat with respect to whether they saw the more N-terminal, middle, or C-terminal portions of these regions. The sequence of residues 13-22 was recognized by three MAbs. The C-terminal region from residues 84-92 was recognized by three MAbs. All MAbs recognized conformational epitopes in that they reacted with two or more regions. Three MAbs (two against free alpha and one against human CG) have linear amino acid sequences as part of their conformational epitope.

The glycoprotein hormones (LH, hCG, FSH, and TSH) have a common 92-amino acid alpha-subunit which is noncovalently linked to a hormone-specific beta-subunit. Synthetic peptides of the alpha-subunit have been shown to inhibit binding of [125I]iodo-hCG to rat ovarian membrane and [125I]iodo-TSH to human thyroid membrane preparations. Synthetic overlapping peptides of the alpha-subunit of hCG were prepared by solid phase techniques and tested in a standard in vitro rat Leydig cell bioassay. Three regions in the alpha-subunit (alpha 1-15, alpha 30-45, and alpha 71-85) were found to stimulate testosterone production. All three regions correlate with inhibition of hCG binding to ovarian receptors, but subtle differences exist between the binding sites and effector sites. These data indicate that the glycoprotein alpha-subunit has intrinsic bioactivity.

Several regions on both the alpha- and beta-subunits of human LH comprise the receptor-binding domain of the hormone. One of these, a disulfide loop peptide containing residues 38-57 on the beta-subunit, also stimulated steroidogenesis in rat Leydig cells. Circular dichroism analysis and a Schiffer-Edmundson helical wheel projection of beta-(38-57) revealed the possibility of an amphipathic alpha-helical structure through its N-terminal region. Secondary structure prediction algorithms do not predict alpha-helix in beta-(38-57), but, rather, suggest a sheet-coil-sheet topology. Homology searches between this peptide and proteins with known structure revealed that the two best matches are with prealbumin-(10-30) and melittin-(1-26). Based on hydrophobic moment calculations, we suggest that beta-(38-57) more closely resembles melittin, a known example of an amphipathic helix. Molecular models were constructed that included an alpha-helix between Pro-39 and Pro-50 producing a hydrophilic face involving Thr-40, Arg-43, and Gin-46. Loop closure was performed either visually or by an incremental minimization procedure, using distance constraints to patch in a disulfide bond. Molecular dynamics at 300, 360, and 1000 K were used to explore the local conformational space, and dynamic structures were minimized. The most reasonable structures were found with the 300 and 360 K simulations, with those at 360 K consistently producing structures with lower conformational energies. In each of these simulations, the N-terminus of the alpha-helix unraveled to form a reverse turn (predicted by the GOR algorithm) which include Cys-38, Pro-39, Thr-40, and Met-41. Simulations at 1000 K produced the most variation in structure, but these were deemed unreasonable. Although not all possible conformations were explored, several models were found that comply with the assumption of an amphipathic helix in the N-terminal half of the peptide.

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.

Eight monoclonal antibodies, specific for the glycoprotein hormone alpha-subunit, were raised against human free alpha-subunit, human FSH, or human CG. All of these antihuman monoclonal antibodies were tested for cross-reactivity with alpha-subunits derived from bovine, porcine, equine, bull frog, sea turtle, turkey, and ostrich glycoprotein hormones. All showed cross-reactivity with affinities ranging from 10(-4) to 10(-8) depending upon the antibody and the species of alpha-subunit. Cyanogen bromide fragments of bovine and equine alpha, when tested with selected antibodies indicated that antigenic determinants could be localized in two regions: alpha 9-33 and alpha 76-92. Comparison of amino acid sequences, and relative potencies, suggest that major antigenic determinants involve residues 21, 22, and 23 (F-F-S in human alpha) and 76-85 (G-G-F-K-V-E-N-H-T-A in human alpha). As part of this study the N-terminal amino acid sequences of bull frog, sea turtle, turkey, and ostrich alpha-subunits were determined and reported for the first time.

The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.

The intercysteine "loop" sequence 38-57 in the beta subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG) [Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, T., Johnson, L., & Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2038]. Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, we have used analogues of hLH beta-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. "Far"-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% alpha-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. Other analogues showed a requirement for a neutral residue at position 47, also highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Cottage cheese (100g) provides only 6% of the calcium Recommended Daily Allowance for adults. Samples of fresh cheese were supplemented with calcium salts (chloride, lactate, or phosphate monobasic) to double the calcium content of the cheese to approximately 145mg/100g, and the effects of the supplementation on taste and microbial stability were studied. Supplementations did not affect pH or microbial stability during 21 d of refrigerated storage. Sensory evaluation results of a 12-member taste panel showed higher flavor and preference scores for control than for all supplemented samples (P<.05) but no difference in odor, color, or texture. Overall, chloride appeared to be preferable for calcium content supplementation because only .26% (wt/wt) of the salt was required to obtain the desired calcium content level and the mineral was evenly distributed in the cheese.

Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.

Fourteen healthy young women were studied through a control and a treatment menstrual cycle in two series of experiments. In the first series, they were given one of four doses of deglycosylated human chorionic gonadotropin (hCG) as a 24-hour infusion during the mid-luteal phase of the cycle. In these studies, there were no significant alterations of the length of the luteal phase of the treatment cycle, and there was no decrease in serum progesterone (P) during the infusion. In fact, serum P increased during the infusion. In the second series of studies, five subjects were given a 48-hour infusion of normal saline during the control cycle, and a 48-hour infusion of deglycosylated alpha-intact beta-hCG during the treatment cycle, both being administered during the mid-luteal phase. Treatment did not alter luteal phase duration and, again, increased serum P. It is concluded that deglycosylated preparations of hCG are not clinically useful as luteinizing hormone antagonists, probably because of residual agonist activity.

Two inhibitors of FSH binding to receptor have been isolated from porcine follicular fluid and shown to have in vitro biological activity. These inhibitors were distinct separable entities with opposite biological effects (agonist and antagonist) on cultured FSH-responsive Sertoli cells. In light of the fact that the agonist-containing fraction (P4) inhibited [125I]human (h) FSH binding to anti-hFSH antiserum as well as to receptor, characterization of this factor was undertaken to determine its relationship to pituitary FSH. The P4 fraction was further purified by affinity chromatography, which removed a major protein from immunoreactive components. Western blotting of sodium dodecyl sulfate-polyacrylamide gels using polyclonal (anti-hFSH) and monoclonal (anti-hFSH beta) antibodies revealed a major immunoreactive band at 55,000 mol wt (Mr). When electrophoresed under reducing conditions, major immunoreactive proteins at 58,000 and 45,000 Mr were identified. These bands were also observed in extracts from bovine testes and raw porcine follicular fluid after electrophoresis and Western blotting. Whereas the monoclonal antibody used to characterize this inhibitor does not recognize porcine pituitary FSH, the Mr of the immunoreactive proteins are greater than that of pituitary FSH, and the immunoreactive bands do not reduce to subunits, as observed for pituitary FSH under reducing conditions, we conclude that gonadal extracts contain FSH-immunoreactive proteins that are immunologically and biochemically distinguishable from pituitary FSH. While the physiological role of these proteins remains to be determined, their presence in gonadal extracts or fluids vitiates assessment of FSH within the gonad by RIA using antiserum against hFSH.

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. We prepared a series of peptides representing the intercysteine "loop" sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) beta subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG beta-(38-57) and hLH beta-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 X 10(-4) and 2.03 X 10(-5) M, respectively, while other peptide hormones and fragments from elsewhere in the beta subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 X 10(-5) M (hCG) and 2.18 X 10(-5) M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG beta-(38-57) peptide, native hCG and beta subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. Helical-wheel projection predicted an amphipathic region in the N-terminal portion of the 38-57 sequence, and circular dichroic measurements showed an increase in ordered structure, especially alpha-helix, when the 38-57 peptides were transferred from an aqueous to a more lipophilic (90% trifluoroethanol) environment. These results indicate that the 38-57 region of beta subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.

The gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) of pituitary origin, and chorionic gonadotropin (hCG, eCG) of placental origin, along with thyroid-stimulating hormone (TSH) constitute a family of glycoprotein hormones. The α-subunit is common to all of the hormones and shows considerable homology from one species to another. The β-subunits are unique for LH, FSH, and TSH. The ß-subunits of hLH and hCG are highly homologous (85%) through the first 114 residues, but hCG β differs in that it has a C-terminus extension rich in serine and proline. This offers a chemical basis for their common biological activities. Baboon chorionic gonadotropin, and eLH and chorionic gonadotropin ß-subunits also have C-terminal extensions. The glycoprotein hormone ß-subunits also show regions of homology between themselves and among different species. A wide range of chemical and enzymatic modifications have been employed in efforts to define residues and sequences in the ß-subunits that may be essential for receptor binding. In general, chemical modifications of reactive amino acids appear to be more tolerated in the ß- than in the α-subunits.

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.