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The immunological properties of a synthetic peptide comprising the carboxyl-terminal 53--84 region of human parathyroid hormone (PTH) have been studied. The immunoreactivity of the synthetic human PTH-(53--84) peptide paralleled that of a 53--84 fragment of the native human hormone prepared by enzymic digestion, in both a standard radioimmunoassay, which was not region-specific, and also a radioimmunoassay specific for the carboxyl-terminal region of PTH. However, in both types of radioimmunoassay the synthetic human PTH-(53--84) peptide was four to five times more reactive than the native human PTH-(53--84) fragment.

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.

The sequence of the amino-terminal portion of human parathyroid hormone, particularly the identity of residues 22, 28, and 30 (the subject of discrepancies in recent published reports), has been reexamined by two basic methods of structural analysis. A fresh lot of human parathyroid hormone isolated from pooled adenoma tissue was analyzed by Edman degradation with identification of critical residues by thin-layer chromatography and gas-liquid chromatography. In the second approach, -14C or tritiated amino acids were incorporated during biosynthesis of the human hormone in slices of parathyroid glands in vitro; the appropriate amino acid residues were then determined as the -14C or tritiated phenythiohydantoin derivatives of the amino acid after Edman degradation, or by peptide isolation after appropriate cleavage with endopeptidase, or both. The results confirm our previous findings that residue 22 is glutamic acid, residue 28 is leucine, and residue 30 is aspartic acid.

cDNA analyses predict that the human calcitonin gene encodes the two precursor proteins of calcitonin and calcitonin gene-related peptide (CGRP). The mRNAs for the putative precursors of these peptides are derived from alternative processing of the primary transcript of this gene. The 75 amino-terminal residues of each preprohormone are predicted to be identical. We have developed the TT human medullary thyroid carcinoma cell line as a model to study human calcitonin gene expression. Mature calcitonin and CGRP are major secretory products of this cell line. Extracts of TT cells radiolabeled with amino acids for 1 h contained only one peak of either immunoprecipitable calcitonin or CGRP on reverse-phase high-pressure liquid chromatography. Each of these species had an Mr = 12,800 as estimated by gel filtration chromatography. The results of partial amino-terminal microsequencing of these two precursors were identical. Residues 2, 11, and 14 of each precursor were proline, and residues 7, 17, 24, 25, 26, and 29 were leucine. The unique alignment of the positions of these amino acids with the cDNA-predicted sequence for the common region of preprocalcitonin and preproCGRP suggests that the site of signal sequence cleavage occurs after residue 25 of both preprophormones. These studies represent the first identification of procalcitonin (116 amino acids) from a human cell line and of proCGRP (103 amino acids) from any tissue.

Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

The beta subunit of human choriogonadotropin (hCG) was previously shown to be phosphorylated by beef skeletal muscle cAMP-dependent protein kinase (Keutmann, H. T., Ratanabanangkoon, K., Pierce, M. W., Kitzmann, K., and Ryan, R. J. (1983) J. Biol. Chem. 258, 14522-14527). The phosphorylation site was primarily at Thr 97, located within the "determinant loop" region proposed by Ward and Moore (Ward, D. N., and Moore, W. T. (1979) Animal Models for Research in Fertility and Contraception (Alexander, N. J., ed) pp. 151-164, Harper and Row, Baltimore, MD) to be important for hormonal activity and specificity. Biological and immunological studies were carried out to determine the effect of this modification on the activity of hCG. The phosphorylated hCG beta recombined with hCG alpha to form phosphorylated hCG (*hCG). The recombined *hCG retained full immunological activity in a radioimmunoassay using anti-hCG serum and 125I-hCG. The biological activities of the *hCG on appropriate rat gonadal cells, as studied by hCG radioreceptor assay, follitropin radioreceptor assay, and adenylate cyclase assay, were 0.29 +/- 0.04, 0.29 +/- 0.07, and 0.69 +/- 0.13 times as potent as hCG, respectively. The reduced receptor binding activity of *hCG was not due to dissociation of the hormone into its subunits. The far ultraviolet CD spectrum of the *hCG showed no gross conformational change induced by phosphorylation. We conclude the following: 1) Thr 97 is not involved in subunit-subunit interaction and is not part of the hCG antigenic site recognized by this particular antiserum; 2) Thr 97 does appear to participate in the hCG-receptor interactions; 3) modification of Thr 97 does not result in an enhancement of follitropin-like activity.

Structural studies have substantiated the concept that the glycoprotein hormones consist of a "common" alpha subunit and "hormone-specific" beta subunit. Despite this, consensus is still lacking concerning certain portions of the amino acid sequences, including alignment of residues 81--82 in the alpha subunit. We have carried out sequence analysis of the alpha subunit of human luteinizing hormone (hLH) to clarify the assignment of these residues and to examine further the nature of the amino-terminal heterogeneity found among the different alpha subunits. Our structure showed residues 80--84 to be -His-Cys-Ser-Thy-Cys-, consistent with findings of others for human follicle stimulating hormone (hFSH) and human chorionic gonadotropin (hCG). The extensive degree of heterogeneity found in the amino terminal region of hFSH and hCG is present to only a minor extent in hLH. The differences in the pattern of amino-terminal heterogeneity among the various hormones may result from differences in the nature of cleavage of subunit from a larger intracellular precursor peptide.

The sequence of the amino-terminal 37 residues of human parathyroid hormone has been established. The hormone used in these studies was isolated in highly purified form from parathyroid adenomata and was subjected to automated degradation in a Beckman sequencer. A high-sensitivity sequencing procedure employing (35)S-labeled phenylisothiocyanate of high specific activity as the coupling agent was used. The sequence obtained differs from that of bovine parathyroid hormone in three of the first 37 positions, and from that of porcine parathyroid hormone in two positions. A single human-specific residue was found (asparagine 16). The sequence obtained differs at three positions (22, 28, and 30) from the structure for human parathyroid hormone reported recently by Brewer et al. [(1972) Proc. Nat. Acad. Sci. USA 69, 3585-3588] and synthesized by Andreatta et al. [(1973) Helv. Chim. Acta, 56, 470-473] We have carefully reviewed our data, reported here in detail, on the sequence positions in dispute. We must conclude, on the basis of all available data, that the structure that we propose is the correct structure. The objective resolution of these discrepancies in structural analysis through further chemical and immunochemical studies is important, since synthesis of human parathyroid hormone, in which there is widespread interest for physiological and clinical studies, must be based on the correct sequence of the human hormone if the peptide is to be genuinely useful.

Using a method for continuous removal of cisternal cerebrospinal fluid (CSF) from freely moving cats, we delineated the circadian nature of the daily rhythm in CSF arginine vasopressin. The daily melatonin rhythm was also monitored in CSF as another marker of circadian function. Under diurnal lighting conditions, both hormones exhibited prominent daily rhythms; the CSF vasopressin rhythm was characterized by high daytime values, whereas the CSF melatonin rhythm was characterized by high nighttime levels. In contrast, drinking behavior exhibited a 24-h component in only one of four animals studied. Daily CSF rhythms of vasopressin and melatonin persisted for over 78 days of study in constant light. The vasopressin rhythm clearly free-ran in this environment, manifesting cycle lengths of slightly greater than 24 h. The daily melatonin pattern split into several components with increasing time in constant light. An acute 8-h phase delay in the daily light-dark cycle resulted in corresponding but gradual phase shifts in both rhythms. These results indicate that both the vasopressin and melatonin rhythms in cat CSF are endogenously generated and are entrained by the daily light-dark cycle.

Vitamin D-deficient rats subjected to thyroparathyroidectomy (TPTX) were used to evaluate in vivo the biological properties of native bovine parathyroid hormone (bPTH) and chemically synthesized fragments and analogues of the hormone on several parameters of hormone action: calcium and phosphorus fluxes, generation of cyclic adenosine 3',5'-monophosphate (cAMP), and the metabolism of 25-hydroxyvitamin D3 [25(OH)D3]. Vitamin D-deficient rats, after TPTX or sham operation, were intravenously infused with a nutrient containing 7.5 mM CaCl2 for 30 h. During the last 7 h, PTH or one of its analogues was infused intravenously at rates between 0.04 and 20 nmol/h. One hour after the start of the peptide infusion, tritiated 25(OH)D3 was injected. Urine was collected hourly for phosphate and cAMP determinations and, at the end of the experiment, blood was obtained to determine the relative accumulation of tritiated 1,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3). Infusion of bPTH-(1--84), bPTH-(1--34), human (h)PTH-(1--34), or [Nle8, Nle18, Tyr34]bPTH-(1--34) amide was accompanied by a comparable dose-dependent decrease in plasma phosphate and a dose-dependent increase in plasma calcium and [3H]-1,25(OH)2D3, and urinary excretion of phosphate and cAMP. An evaluation of [Nle8, Nle18, Tyr34]bPTH-(3--34) amide, a potent inhibitor of PTH action in vitro in the renal adenylate cyclase assay, revealed that the analogue possessed weak agonist properties in vivo. The analogue increased excretion of both cAMP and phosphate in the urine, decreased plasma phosphate levels, and increased the accumulation of [3H]-1,25(OH)2D3 in the plasma. This multiparameter model system should aid in the elucidation of the in vivo biological effects of PTH and its analogues.

We have recently proposed that the major secreted isoprotein form of human apolipoprotein A-I (designated apo A-I2) is modified extracellularly to become the predominant apo A-I form seen in plasma (designated apo A-I4). In the current report we demonstrate that the primary translation product of human apo A-I (designated apo A-I2p) has a 24-amino-acid NH2-terminal extension with a sequence of Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe- Leu-Thr-Gly-Ser-Gln-Ala-Arg-His-Phe-Trp-Gln-Gln. The first 18 amino acids of this NH2-terminal extension are cleaved intracellularly by the signal peptidase, resulting in the formation of apo A-I2, which is the secreted form of apo A-I. Sequence analysis of apo A-I2 confirmed that it contains a hexapeptide extension at its NH2 terminus compared to apo A-I4. This observation demonstrates that apo A-I2 is a propeptide and that the apo A-I2 to apo A-I4 conversion involves the removal of the NH2-terminal hexapeptide of apo A-I2 by a protease in plasma, lymph, or both. Our findings indicate that apo A-I is synthesized as a prepropeptide, which undergoes intracellular and extracellular proteolysis to attain the major plasma apo A-I4 isoprotein form.

The amino acid sequences of two previously uncharacterized regions of the mouse anterior pituitary common precursor to adrenocorticotropin (ACTH) and beta-endorphin (pro-ACTH/endorphin) were determined. Portions of the NH2-terminal region of pro-ACTH/endorphin (called the 16K fragment) and the region between ACTH and beta-endorphin (called gamma-lipotropin) were sequenced by Edman degradations of biosynthetically labeled immunoprecipitated proteins and by Edman degradations of purified 16K fragment and beta-lipotropin. With a combination of these two approaches, 29 of the first 34 residues at the NH2-terminal end of the mouse 16K fragment were determined. The NH2-terminal region of the mouse 16K fragment was found to be nearly identical with the homologous porcine and bovine molecules. The complete amino acid sequence of the NH2-terminal region of gamma-lipotropin was determined. In contrast to the highly conserved nature of the 16K fragment, mouse gamma-lipotropin was found to differ substantially from the gamma-lipotropins of other species. Although the NH2-terminal and beta-melanotropin-like regions of the mouse gamma-lipotropin are similar to the corresponding regions of other gamma-lipotropins, the intervening region of mouse gamma-lipotropin is substantially shorter than it is in other gamma-lipotropins. In addition, mouse gamma-lipotropin lacks the pair of basic amino acids that normally mark the proteolytic cleavage site used to produce beta-melanotropin from gamma-lipotropin.

During the isolation of human parathyroid hormone there is an extensive loss of immuno-assayble hormone over the successive extraction steps, due in part to the presence of fragments that are soluble in 4% trichloroacetic acid. These fragments are derived from both the amino- and carboxyl-terminal regions of the hormone. The hormonal fractions precipitated with trichloroacetic acid were further purified by gel filtration and ion-exchange chromatography. At the final ion-exchange purification step, some preparations of the hormone eluted in multiple fractions. When the various components were characterized separately by immunoassay, amino acid composition, enzymic cleavage and partial sequence analysis, they were found to be closely comparable, although the most acidic fraction contained a blocked terminal amino group. Extraction of a number of batches of tissue permitted revision of the amino acid composition of human parathyroid hormone. Biosynthetic studies with labelled amino acids confirmed the absence of tyrosine and the presence of phenylalanine and threonine and localized these residues to definite regions of the molecule.

1. The present paper describes a detailed study of the specificity of high-affinity antibodies obtained from the yolk of eggs laid by a chicken successfully immunized with synthetic human parathyroid hormone (hPTH)-(1-34). 2. Using 125I-labelled bovine parathyroid hormone (bPTH)-(1-84) purified by high performance liquid chromatography (HPLC) as tracer, and hPTH-(1-34) as reference, we found superimposable curves with hPTH-(13-34), bPTH-(13-34) and bPTH-(1-34); the [Asp-76]hPTH-(1-84) peptide showed a molar percent cross-reactivity (calculated at 50% B/BO) of 73% and the bovine sequence 1-84 of 15%. 3. Studying amidated [Tyr-34] bovine PTH fragments we found the highest cross-reactivity with the peptide 7-34 (33%) followed by 20-34 (1.7%) and 25-34 (0.4%). The bovine sequence 1-25 showed a low reactivity (0.7%) and the 1-12 sequence none at all. Rat parathyroid hormone (rPTH)-(1-34) showed low cross-reactivity (3.1%), the same occurring with peptide [Tyr-34, Norleu-8,18] bPTH-(1-34) (1.5%). 4. The data suggest that the antibodies studied recognize epitopes in or near amino acids 18 to 25 of the sequence of the hPTH molecule. This region of the molecule is coincident with the antigenic determinant predicted by the analysis of the hydrophilicity plot of the hPTH-(1-34) peptide.

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.

We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH alpha-subunit, with RCXM-alpha as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-alpha. A tryptic digest of RCXM alpha-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM alpha-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency(panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-alpha were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of alpha-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit.

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)

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.

Fresh frozen bovine parathyroid glands were defatted in acetone, when extracted with phenol. Following trichloroacetic acid precipitation, the resultant peptides were chromatographed on Sephadex G-100. Parathyroid hormone (BPTH) characteristically elutes in the fourth peak. However, we also observed significant hormonal activity, both biological and immunological, in the fifth elution peak. The peak V material had potent hypercalcemic activity in the rat and chick, and stimulated adenylate cyclase activity in the rat renal cortex bioassay. This material was further purified by ion exchange chromatography on carboxymethylcellulose in 8 M urea. The biological activity of the purified peptide (3700 MRC units/mg) was equivalent to that of the native hormone on a molar basis. Amino acid analysis, carboxypeptidase digestion, and partial Edman sequence analysis identified this material as BPTH 1-65, a hormonal fragment lacking the C-terminal 19 residues of the 84 residue hormone molecule. Several immunoassays using different anti-PTH antisera had variable reactivity toward the BPTH 1-65 fragment, showing that it may be useful for further characterizing antibody recognition sites. The presence of a lysine residue at position 65 suggests a tryptic-like cleavage may be responsible for the genesis of this hormonal fragment. Further investigation will be necessary to determine if this peptide has physiological significance.

[32P]ATP-citrate lyase phosphorylated by the cAMP-dependent protein kinase was partially digested by trypsin. Two tryptic 32P-labeled phosphopeptides containing more than 90% of the 32P radioactivity present on the phosphorylated enzyme were purified and found to have overlapping amino acid sequences around the same phosphorylated site (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg). Tryptic digestion of 32P-labeled ATP-citrate lyase purified from 32P-labeled hepatocytes exposed to glucagon yielded a major 32P-labeled peptide of identical amino acid composition with that indicated above. Thus, the site on ATP-citrate lyase phosphorylated by the cAMP-dependent protein kinase in vitro resides on the same octapeptide as the site of glucagon-stimulated phosphorylation in intact hepatocytes.

Removal of long-chain asparagine-linked carbohydrates leads to loss of receptor-effector coupling in gonadotropin target tissue. This implies a direct interaction between carbohydrate and cell membrane components. To examine other mechanisms by which carbohydrate could activate post-receptor events, we have used sequence-specific and conformation-specific alpha-subunit radioimmunoassays as probes for conformational changes in deglycosylated choriogonadotropin and follitropin. Immunoreactivity of either hormone was enhanced 8-13-fold after removal of carbohydrate by anhydrous hydrogen fluoride. Removal of sialic acid alone had little effect on reactivity. Based on the specificity of the antisera, the effect could be localized to a region in the amino-terminus remote in the linear sequence from actual sites of carbohydrate attachment. The results suggest that a conformational change in the alpha-subunit could account, at least in part, for the observed effects of deglycosylation on glycoprotein hormone action.

Peptides prepared by solid-phase synthesis during a systematic study of structure-activity relations in parathyroid hormone have been subjected, after sequential purifications, to rigorous analysis of purity by a variety of analytical techniques including sequence analysis by the Edman procedure. The present paper undertakes a critical appraisal of the utility of different tests of peptide purity and the use of the procedures in guiding and monitoring optimal synthesis strategies. Sequence analysis of a peptide representing the amino-terminal 34 residues of bovine parathyroid hormone (bPTH-(1-34)) revealed the presence of at least 30% of contaminating error peptides which were undetected by other analytical procedures. The major contaminant was identified as a peptid in which glutamine at position 29 was deleted. A repeat synthesis using fluorescamine rather than ninhydrin to monitor the coupling reaction resulted in a preparation that lacked the contaminant resulting from deletion. These findings demonstrate the particular value of sequence analysis in the evaluation of purity of peptides synthesized by the solid-phase technique.

Proparathyroid hormone (calcemic fraction-A) is a biosynthetic precursor of parathyroid hormone in bovine glands. Limited amounts of the isolated prohormone have been obtained for the purpose of initial structural studies. The results of automated sequence analysis on two separate preparations indicate that the N-terminal region of the prohormone consists of the sequence Lys-Ser-Val-Lys-Lys-Arg followed by a sequence exactly corresponding to residues 1 to 34 of bovine parathyroid hormone. Also observed was a minor sequence (about 12% of the total) in which the N-terminal lysine was absent. These data suggest that more than one species of prohormone may exist. Due to the small quantities of sample available, the analyses were restricted to the N-terminal portion of the prohormone only. However, because the amino-acid composition of the prohormone indicates it to be a molecule containing more than 100 amino acids, the possibility remains that additional residues occur at the C-terminus. Thus, the prohormone structure based on these data is believed to consist of the hexapeptide sequence above, followed by the known sequence of the 84 residues in parathyroid hormone, possibly followed by an additional sequence of 10-15 residues.

The alpha and beta subunits of porcine FSH were isolated and purified to homogeneity as judged by SDS disc gel electrophoresis. The isolated subunits had less than 1% of the biological activity of the native hormone but were capable of recombining to generate at least 23% of the activity of the native hormone. The molecular weights calculated from hydrodynamic properties were 12,600 for pFSH alpha and 17,200 for pFSH beta. Total carbohydrate (g/100 g) was 18.9 for alpha and 15.1 for beta. The sialic acid content of alpha (3.8 g/100 g) exceeded that of beta (0.2 g/100 g). The alpha subunit contained significantly more lysine, alanine phenylalanine and methionine and significantly less aspartic acid, threonine, valine, isoleucine, leucine and tryptophan than the beta subunit. Evidence was found for N-terminal heterogeneity and an internal cleavage in the isolated alpha subunit.

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.

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.

We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.

Human chorionic gonadotropin (hCG) and its subunits were studied as substrates for cAMP-dependent protein kinase. Phosphorylated residues were identified in peptide fragments by sequence analysis following appropriate hydrolysis and purification. Only the free beta-subunit was phosphorylated. Intact beta subunit incorporated 1 mol of phosphate/mol of peptide. This was localized to Thr 97, within the sequence -Arg-Arg-Ser-Thr-, which resembles one type of acceptor site for cAMP-dependent phosphorylation. Since beta Thr 97 did not phosphorylate in native hCG, this residue may be masked by the alpha subunit. Reduced and carboxymethylated hCG beta phosphorylated to 2.8 mol of phosphate/mol of peptide. Besides Thr 97, phosphate was also found at Ser 66 and Ser 96. Ser 66 occurs within a segment recognized to be favored for phoshorylation with the general sequence -Arg-X-X-Arg-X-X-Ser-. The appearance of this site in the linearized peptide suggests that Ser 66 is "buried" in the native conformation of the beta subunit. Two synthetic peptides representing residues 93-102 of hCG beta were also examined. The disulfide form of the peptide phosphorylated at Thr 97 while the linear peptide phosphorylated at Ser 96. Conformation as well as primary structure can thus influence the site of phosphate incorporation.

Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.

The rat PTH molecule contains five sequence differences from either the bovine or the human hormone within the biologically active 1-34 region. A synthetic rat 1-34 peptide was tested for activity by in vitro activation of canine and rat renal adenylate cyclase and binding to canine renal membrane receptors. The mean potency of 21,400 Medical Research Council units/mg in the canine adenylate cyclase system and 24,900 in the rat system was 8- to 10-fold higher than human 1-34 and 2- to 4-fold greater than bovine 1-34. These values represent the highest potency we have observed to date for a PTH preparation by these assay systems. In contrast, receptor binding of the rat fragment was comparable to that of bovine and human 1-34. Half-maximal inhibition of radioligand binding occurred at 1.7- 2.0 X 10(-9) M with all synthetic hormones. Hence, the amino acid substitutions in rat 1-34 appear to affect the cyclase-activating sequence domain without increasing avidity for the receptor. Analogs combining the rat sequence with modifications known to enhance receptor binding and/or retard enzymatic degradation offer a promising approach to the preparation of still more potent parathyroid agonists.

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.

32P-labeled ATP-citrate lyase isolated from 32P-labeled hepatocytes treated with insulin contained 1.6-1.8-fold greater 32P-radioactivity per mg protein than control enzyme. Both enzyme preparations were digested in parallel with trypsin until 94% of all 32P-radioactivity was rendered acid soluble. Quantitative high performance liquid chromatographic peptide mapping of the tryptic digests revealed a principal 32P-peptide which accounted for at least 80% of the insulin induced increment in 32P-radioactivity of native lyase. This peptide was purified, sequenced, and the site of 32P-phosphorylation assigned by two methods: electrophoresis (pH 6.5) of residual peptide after each step of Edman degradation and solid phase sequencing. The site of insulin-directed phosphorylation of ATP-citrate lyase (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg) is the same as that directed by glucagon, and, in turn, identical with that phosphorylated by the cAMP-dependent protein kinase in vitro.

The present study was conducted to show that the hypercalcemic and the vascular relaxing activities of PTH are two separable properties. During the hypotensive action of the synthetic fragment bovine (b) PTH-(1-34), plasma calcium levels were not significantly changed. Mild oxidation with hydrogen peroxide abolished the hypotensive and hypercalcemic actions of bPTH-(1-84). However, the same treatment on bPTH-(1-34) abolished only the hypotensive and not the hypercalcemic action. Analysis of the amino acid composition revealed only the oxidation of the methionines to methionine sulfoxides. The other amino acids remained unchanged. In addition, the analog with methionines replaced by norleucine, [Nle8,Nle18,Tyr34]bPTH-(1-34), was active in all the vascular assays, and these activities were unaffected by hydrogen peroxide treatment of the molecule. Perhaps the methionine sulfoxides in the hydrogen peroxide-treated bPTH-(1-34) affected the changes of the molecule in such a manner that the part of the molecule for the vascular action but not that for the hypercalcemic action was no longer accessible to the receptors of the target organs. The hypotensive pentapeptide, bPTH-(24-28), was not active in the hypercalcemic assay. All these data are consistent with our hypothesis that the vascular relaxing and the hypercalcemic actions of PTH are two separate properties of the molecule.

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.

TSH is a glycoprotein hormone whose carbohydrate content varies among different species. Although recent studies suggest that variants of TSH deficient in carbohydrate occur naturally, the significance of the carbohydrate moiety of TSH in respect to its thyrotropic function is unclear. The present studies were undertaken, therefore, to examine this question. A highly purified preparation of bovine TSH (bTSH) was deglycosylated by treatment with anhydrous hydrogen fluoride. Amino acid and carbohydrate analyses of the original and deglycosylated preparations indicated that approximately 85% of the carbohydrate originally present had been removed and that the protein moiety was unaltered. As judged from TSH radioreceptor assays, bTSH and deglycosylated bTSH (dg-bTSH) bound to human thyroid membranes with equal affinity, since both caused a half-maximal inhibition of [125I]bTSH binding at approximately equal concentrations. Nonetheless, dg-bTSH at optimal concentration displayed only about one third the activity of intact TSH in stimulating adenylate cyclase activity in human thyroid membranes. dg-bTSH also antagonized the adenylate cyclase-stimulating activity of intact bTSH in this system, but only weakly, since abolition of the bTSH effect required an approximately 40-fold higher concentration of dg-bTSH. In cultures of FRTL5 cells, a cloned line of follicular cells derived from normal rat thyroid, both intact and dg-bTSH enhanced cell growth, as measured by [3H]thymidine incorporation and stimulated cAMP release in the medium, but the response elicited by dg-bTSH was much less than that caused by equal concentrations of the intact hormone. In accord with the findings in the in vitro assays, dg-bTSH evoked a much smaller response than bTSH did in the in vivo mouse assay. It is concluded that although not required for receptor recognition, the carbohydrate moiety of bTSH is essential for the full expression of its biological activity.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.