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L C Giudice

Publications and source records attributed to L C Giudice.

122 records · Page 7Linked to original sources
PubMed1979

Comparison of bovine and mouse pituitary glycoprotein hormone pre-alpha subunits synthesized in vitro.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.

Amino Acid Sequence↗
PubMed1979

Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.

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↗
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↗
PubMed1976

Structure and structure-function relationships in glycoprotein hormones.

Relationships between the sequences of thyrotropin (thyroid-stimulating hormone, TSH), lutropin (luteinizing hormone, LH), human choriogonadotropin (chorionic gonadotropin, hCG) and follitropin (follicle-stimulating hormone, FSH) are now well established. Each beta-subunit contains six disulphide bonds and considerable homology is seen when all four linear sequences are aligned with half-cystine residues in juxtaposition. Major questions about the tertiary structures of the subunits and their interactions to form active hormone remain. Determination of the disulphide bridges in both alpha- and beta-subunits has not yielded to usual methods and conflicting data about the alpha-subunit have been reported. Partial reduction of the beta-subunits of LH and TSH with subsequent labelling of the cysteines formed has shown that a single bond is first reduced. This bond is between positions 93 and 100 in LH-beta and the corresponding positions 88-95 in TSH-beta. Thus, as would be expected from the fact that interhormone hybrids can be made with the common alpha-subunits, the chemical data, though still limited, indicate similar tertiary structures for the different beta-subunits. To investigate whether other useful intermediates can be obtained after partial reduction, we have studied reduction and derivative formation in various conditions. Intact LH is more resistant to reduction than either its alpha- or beta-subunit but no intermediates have been observed which are not present after partial reduction of individual subunits. Preliminary experiments on the reoxidation of fully reduced alpha-subunit show that the reoxidized material will recombine with native beta-subunits to yield biologically active TSH or LH. Studies from this and other laboratories on chemical modifications of several amino acid residues of glycoprotein hormones and their subunits are also summarized.

Amino Acid Sequence↗
PubMedDate not supplied

Insulin-like growth factor family in Graafian follicle development and function.

The insulin-like growth factor (IGF) family plays an important role in follicle development, dominant follicle growth and steroidogenesis, and follicular atresia. Insulin-like growth factor (IGF)-II is the primary IGF in human ovary, acting as a mediator of gonadotropin action. IGF-II stimulates granulosa steroidogenesis, and its actions, along with those of follicle-stimulating hormone (FSH), are inhibited by IGF binding protein (IGFBP)-4. At the time of follicle selection, in the estrogen-dominant follicle, granulosa IGF-II synthesis increases dramatically. Simultaneously, in the selected follicle, IGFBP-4, an inhibitor of IGF-II action and abundant within androgen-dominant follicles, is proteolyzed by a specific IGFBP-4 protease, resulting in decreased affinity of IGFBP-4 for IGF-II. The IGFBP-4 protease has recently been identified in human ovary as pregnancy-associated plasma protein-A (PAPP-A). In the Graafian follicle, IGF-II bioavailability is increased to act as a mediator of FSH action, by increased synthesis of this peptide and concomitant decrease in an inhibitor of its actions by proteolysis of IGFBP-4.

Animals↗
PubMedDate not supplied

Role of growth hormone in ovarian physiology and onset of puberty.

OBJECTIVE: To review the role of growth hormone (GH) in the timing of puberty and to summarize the interactions between the somatotrophic and reproductive axes. The use of recombinant GH in patients with GH deficiency is outlined. METHODS: A MEDLINE search was performed of English language manuscripts dealing with the role or use of GH in puberty and reproduction. RESULTS: Growth hormone appears to augment the rate of sexual maturation once a pubertal pattern of gonadotropin secretion is established. Growth hormone may modulate gonadal steroid production by increasing hepatic insulin-like growth factor-I production or by acting directly on GH receptors, localized recently in the granulosa cells and corpus luteum in the human ovary. Patients with isolated GH deficiency exhibit short stature, truncal adiposity, and delayed puberty. However, patients with isolated GH deficiency ultimately undergo spontaneous puberty, and women with GH receptor defects are fertile, suggesting that GH most likely acts as a co-gonadotropin to augment the actions of FSH and LH on estradiol and progesterone production. CONCLUSIONS: Growth hormone appears to increase the rate of sexual maturation once a pubertal pattern of pituitary gonadotropin secretion is established. The ovary is a site of GH reception and action, where it can potentiate steroidogenesis and gametogenesis either directly or through endocrine actions. Continued basic and clinical research is needed to characterize further the role of GH in reproductive physiology.

Animals↗