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J G Pierce

Publications and source records attributed to J G Pierce.

At least 37 records · Page 2Linked to original sources
PubMed1981

Production and release of thyrotropin and its subunits by monolayer cultures containing bovine anterior pituitary cells.

We have developed a dispersed cell monolayer system derived from bovine anterior pituitary glands. Fresh 1- to 6-week-old calf anterior pituitaries were mechanically and enzymatically dispersed and incubated with Dulbecco's Modified Minimal Essential Medium containing 10% hypothyroid goat serum. The media and cell extracts from confluent monolayers were analyzed for bovine TSH and free alpha, and TSH beta subunits by specific homologous RIAs. Basal levels of TSH, free alpha, and free TSH beta subunits in the media were 6.2 +/- 0.3, and 0.95 +/- 0.05 ng/10(6) cells . 24 h, respectively. Hence, an 8- to 10-fold excess of free alpha over free TSH beta subunits was released into the medium. Intracellular basal levels of TSH, free alpha, and free TSH beta subunits were 27.6 +/- 1.7, 10.7 +/- 0.2, and 2.6 +/- 0.3 ng/10(6) cells . 24 h, respectively, and indicated a 3- to 4-fold excess of free alpha over free TSH beta subunits within the cells. The total alpha-subunit to total beta-subunit ratio was 2:1. TRH stimulated release of TSH and its subunits in a dose-dependent fashion, with a half-maximal dose of 2 nM and a maximal response dose of 10 nM. Stimulation with 100 nM TRH increased the levels of TSH, free alpha, and free TSH beta subunits (450-900%, 180-200%, and 300-400%, respectively) in medium, with concomitant decreases within cells. Treatment with thyroid hormones decreased basal and blunted TRH-stimulated levels of TSH and its subunits in medium but had no effect on intracellular stores. However, large doses of T4 (25 nM) or T3 (1 nM) did not completely abolish the TRH (100 nM)-stimulated hormone response. TRH and thyroid hormones affect the release of TSH and TSH beta to a greater extent than they do the alpha-subunit. Finally, total alpha and TSH beta subunit production was increased with TRH stimulation and decreased with thyroid hormone exposure. Thus, an in vitro system to study the net production and secretion of TSH and its subunits in the normal pituitary thyrotrope has been established. (Endocrinology 108: 387, 1981)

Animals↗
PubMed1981

Doubly radioiodinated luteinizing hormone: preparation and characterization.

Bovine [131I]Iodo-alpha LH-[125I]iodo-beta LH (**LH) has been prepared and shown to be physically and biologically equivalent to unmodified hormone. The beta-subunit was modified with 125I, purified by adsorption to Concanavalin A-Sepharose and elution with methylmannoside, added to alpha-subunit, and allowed to reassociate to intact hormone. Iodination with 131I was then carried out in the reassociation mixture and **LH was isolated by gel filtration. Both gel electrophoresis and rechromatography on Sephadex G-100 showed that both radiolabels comigrated with unmodified hormone. Sodium dodecyl sulfate gel electrophoresis showed that 131I was found in the alpha-subunit and 125I in the beta-subunit; this result is in agreement with studies by others which show that the tyrosines of the beta-subunit are nonreactive in intact hormone. In receptor-binding assays, both radiolabels were specifically displaced in a similar fashion by LH. Scatchard analysis showed high affinity binding (Ka approximately equal to 1.5 X 10(10) M-1) for both labels. Comparison of receptor-binding activity with steroidogenic activity showed that iodinated hormone molecules not only bound to receptor but also stimulated testosterone production. The demonstration that full biological activity is retained with iodination in both subunits shows that such doubly labeled LH can be used to monitor the disposition of both subunits simultaneously during interaction of the hormone with target cells.

Animals↗
PubMed1980

Oligosaccharide moieties of glycoprotein hormones: bovine lutropin resists enzymatic deglycosylation because of terminal O-sulfated N-acetylhexosamines.

The oligosaccharides of the bovine pituitary gonadotropin lutropin are N-linked to asparagine residues. These carbohydrates are unusual in that, although they contain the mannose, N-acetylglucosamine, and fucose typical of N-linked oligosaccharides, they also contain one residue of N-acetylgalactosamine but insignificant amounts of sialic acid or galactose. These oligosaccharides exhibit complete resistance to several exoglycosidases. This is in contrast to the ready release of peripheral sugars from human chorionic gonadotropin, a placenta hormone which has oligosaccharides of the complex type with terminal sialic acid and galactose residues. Stability of the lutropin hexosamines to periodate oxidation and reduction (Smith degradation) together with other data show that one residue of N-acetylgalactosamine and one of N-acetylglucosamine are peripheral to two periodate-sensitive mannose residues. The insensitivity to periodate of these two terminal amino sugars is found to result from a sulfate group covalently linked to each; sulfation of these hexosamines is also the most probable reason for the resistance to enzymatic deglycosylation. The alpha subunits of bovine thyrotropin and human pituitary lutropin also contain sulfate, in contrast to human chorionic gonadotropin. The results indicate that sulfating enzymes are present in the pituitary and that sulfation of peripheral sialic acids in the placental gonadotropin. The data lead to a partial structure for the oligosaccharides of bovine LH as follows: (formula see text).

Acetylgalactosamine↗
PubMed1980

Receptor-binding activity of highly purified bovine luteinizing hormone and thyrotropin, and their subunits.

Highly purified preparations of bovine TSH (bTSH) and LH (bLH) and their subunits have been obtained by affinity chromatography using immobilized antibodies directed against counterpart subunits. The purified preparations were assessed for biological activity in radioligand-receptor assays for TSH and LH. After affinity purification against bLH beta, a TSH preparation whose initial potency in the LH assay had been 0.15% that of LH, failed to compete with [125I]LH in amounts up to 100 microgram. Thus, it appears that bTSH does not bind to LH receptors in the rat testis and that interaction of less purified TSH with gonadotropin receptors is attributable to LH contamination. In contrast, LH, whose initial potency in the TSH receptor assay was 0.6% that of TSH, retained a potency of 0.004% of TSH (equivalent to 3.6 mU/mg) after immunoadsorption by anti-bTSH beta. The retention of TSH receptor-binding activity by affinity-purified LH indicates that the LH molecule (like hCG) has a low intrinsic thyroid-stimulating activity. Affinity-purified LH subunits have little or no demonstrable affinity for the LH receptor in vitro. Affinity-purified TSH subunits and affinity-purified LH, however, exhibit very weak receptor-binding activity in the TSH radioligand receptor assay. An evaluation of the capacity of the immunoadsorbents to remove TSH from artificial mixtures suggests that the residual binding does not result entirely from contamination, and therefore, that alpha-subunits as well as LH have some intrinsic TSH-binding activity.

Animals↗
PubMed1979

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.

Amino Acid Sequence↗
PubMed1979

Purification and receptor binding properties of complexes between lutropin and monovalent antibodies against its alpha subunit.

A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its alpha subunit, which is common to the glycoprotein hormones, has been purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex is heterogenous with respect to molecular size; 70--80% of the hormone is complexed with either two or three Fab fragments. The LH-Fab alpha complexes retain only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permits direct measurement of competition between receptor and the bovine complex because the alpha portion of the human hormone does not cross react significantly with antibodies directed against bovine alpha subunits.) Complex formation does not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-beta immunoassay which distinguishes free beta subunit from intact LH. Complexing of LH with Fab-alpha fragments also causes little or no change in the affinity of the hormone's beta subunit for anti-LH-beta antibodies indicating that significant changes in beta subunit conformation did not occur. The data show that at least two well-separated antigenic regions on the alpha subunit are exposed to the surface in the intact hormone. They are also in agreement with the proposal that the loss of binding activity to receptor is due to steric effects rather than to changes in conformation or dissociation, and that there may be sites on the alpha subunit which interact directly with the receptor.

Animals↗
PubMed1978

Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary.

Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).

Animals↗
PubMed1978

Studies on the reduction and reoxidation of the disulfide bonds of the alpha and beta subunits of human choriogonadotropin.

Reoxidation of the disulfide bonds of the alpha-subunit of human choriogonadotropin after their complete reduction yields a product which is indistinguishable from the native subunit in its electrophoretic pattern in polyacrylamide gel and in its ability to recombine with the beta subunit of bovine lutropin. The circular dichroism of reoxidized human choriogonadotropin-alpha is essentially identical to that of the native alpha-subunit, except for slightly more negative ellipticity in the region of 240 mm. Hybrid hormone preparations obtained by recombination of reoxidized or native human choriogonadotropin-alpha with native lutropin-beta exhibit identical electrophoretic patterns in polyacrylamide gels, elution profiles in gel filtration, receptor binding activities, and CD spectra. However, reoxidation of human choriogonadotropin-beta under the same conditions does not yield a product which resembles the native beta subunit in its electrophoretic pattern on gels, its CD spectrum or its ability to recombine with the alpha subunit.

Chorionic Gonadotropin↗
PubMed1978

Effect of conformation of hCG-beta on generation of hCG-specific antibody.

Most antisera generated to isolated highly purified beta subunits of human glycoprotein hormones are not sufficiently sensitive to detect physiologic blood levels of the native hormone. In the dissociated state, beta subunits assume a conformation different from that in the native hormone. Since antisera to alpha subunits have essentially no cross-reactivity between species, highly purified hCG-beta was combined with bTSH-alpha. That hybrid served as immunogen to assess whether sensitive, specific hCG antisera would more likely result than using hCG-beta alone. Of five animals immunized, three developed sufficiently sensitive and specific antisera. The results of these studies strongly suggests that human glycoprotein beta subunits combined with non-human alpha subunit are more likely to yield specific, sensitive antisera than when either isolated beta subunit or the native human glycoprotein hormone, containing common alpha determinants, serves as immunogen.

Animals↗
PubMed1977

Separation of functional and non-functional beta subunits of thyrotropin preparations by polyacrylamide gel electrophoresis.

Electrophoretic patterns of intact human and bovine TSH and bovine LH can be clearly distinguished from those of their subunits in 12% polyacrylamide gels, thus providing an easy method of examining subunit recombination. Two distinct components of both bovine and human TSH-beta subunits are observed, of which only one recombines with alpha subunits. Both beta-components cross-react with antisera to TSH and TSH-beta and have, within experimental error, identical amino acid compositions. Thus, the non-recombining component is a non-functional form of TSH-beta which has retained its immunological specificity, and the data explain why the recovery of biological activity during the recombination of TSH subunits is substantially less than with several other glycoprotein hormone preparations.

Amino Acids↗
PubMed1976

Studies on the disulfide bonds of glycoprotein hormones. Course of reduction of bovine luteinizing hormone, bovine thyroid-stimulating hormone, and their subunits.

The five disulfide bonds of isolated alpha subunits of luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) are completely reduced at pH 8.5 in 15 min with no denaturant required and with only a slight excess of reducing agent. At pH 7.0, reduction is complete after 6 to 10 h. These results together with an earlier study concerning the positions of the two most readily reduced bonds (Cornell J.S., and Pierce, J.G. (1974) J. Biol. Chem. 249, 4166-4174) show that, in the isolated alpha subunit, all disulfides are readily accessible, although it is possible that a change in conformation, after rapid initial reduction of two disulfides, makes the remaining three more susceptible to reduction. No partially reduced and S-carboxymethylated intermediates were found at pH 7.0 other than those seen at pH 8.5, nor were additional intermediates found at pH 8.5 when reduction was initiated in the presence of alkylating agent. In contrast, reduction of the beta, hormone-specific, subunits of LH and TSH, while complete at pH 8.5 after 2 to 6 h, does not proceed to completion at pH 7.0 even after 24 h or upon addition of 6 M urea or large concentrations of reducing agent, and partially reduced intermediates useful in location of disulfide bridges can be trapped (e.g. Reeve, J.R., Cheng, K.-W., and Pierce, J.G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155). Little or no reduction of the intact hormones is found at pH 7.0 in the absence of denaturing agents. This protection by the intact structure shows that the two most readily reduced disulfides of the alpha subunit and the single most readily reduced sidulfide of the beta subunits are either in regions of subunit-subunit contact or that these bonds become more reactive in the isolated subunits because of different influences by neighboring groups. At pH 8.5, intact LH is completely reduced after 6 h, but intact TSH is more resistant to reduction, which may reflect a higher affinity between subunits than exists in LH.

Animals↗
PubMed1976

Studies on the disulfide bonds of glycoprotein hormones. Complete reduction and reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone.

Reoxidation of the disulfide bonds of the alpha subunit of bovine luteinizing hormone (LH) after their complete reduction both in the presence and absence of denaturing agent yields a product which is indistinguishable from the native subunit in its electrophoretic pattern on polyacrylamide gels and in its ability to recombine with the beta subunits of both luteinizing hormone and thyrotropin. The circular dichroism spectrum of the reoxidized alpha subunit is essentially identical to that of native alpha subunit except that its maximum at 233 nm is smaller than observed with native LHalpha. The intact hormone preparations obtained by recombination of reoxidized alpha subunit with native LH-beta exhibit electrophoretic patterns in polyacrylamide gels, elution profiles on gel filtration, binding activities to a membrane fraction from rat testes, and circular dichroism spectra identical to those of native LH and recombinants of native LH-alpha with the beta subunit. Recombinants of native or reoxidized LH-alpha with the beta subunit of thyrotropin are also indistinguishable in their electrophoretic patterns on polyacrylamide gels and in their in vivo activities of stimulating 32P uptake in thyroids of day-old chicks. While this study does not preclude that the alpha subunit may be biosynthesized as part of a larger precursor protein, the data demonstrate that sufficient information is present in the linear sequence of the alpha subunit to allow folding and formation of disulfide bonds to yield a functional alpha subunit.

Animals↗