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1.
Y Saito Y Ishii S Koyama K Tsuji H Yamada T Terai M Kobayashi T Ono M Niwa I Ueda 《Journal of biochemistry》1987,102(1):111-122
The high-level synthesis of alpha-human atrial natriuretic polypeptide hormone in Escherichia coli has been achieved based on the idea that the yield of a small, basic and unstable polypeptide, such as the natriuretic polypeptide, would be improved by fusion with an appropriate protective polypeptide to construct a neutral fused polypeptide. We prepared an expression vector, pCLaHtrp3t, coding a neutral polypeptide containing 130 amino acid residues in which the polypeptide hormone was fused to a newly designed protective polypeptide through lysine as an enzymatically cleavable residue. The fused polypeptide was synthesized at the high level of 32% of total cellular proteins and at 4.7 X 10(6) molecules per single cell. It was recovered as cellular insoluble fraction and purified to homogeneity. For the isolation of the peptide hormone from the resultant fused polypeptide, Achromobacter protease I, a lysine-specific endopeptidase was used, because it has sufficient activity even in 8 M urea. The recombinant natriuretic polypeptide was indistinguishable from native alpha-human atrial natriuretic polypeptide as regards amino acid sequence as well as biological activity. 相似文献
2.
《Journal of Chromatography B: Biomedical Sciences and Applications》1984,305(1):63-71
High-performance liquid chromatography (HPLC) is being used extensively to characterize active polypeptides, precursor processing mechanisms, and cooperative peptide—protein noncovalent complexes in neuroendocrine pathways for neurohypophysial peptide hormones, oxytocin and vasopressin, and the hormone-associated proteins, neurophysins. Reversed-phase and ion-exchange HPLC polypeptide mapping have been used to detect the hormones, associated proteins, and other molecular forms containing these. This mapping but also ultimately to identify anatomical sites which contain the neurophysin/ hormone molecular pathways and to define the relatedness of polypeptide forms contained in different pathways. Reversed-phase HPLC also has provided a means to study proteolytic precursor processing, both to isolate synthetic and semisynthetic polypeptides and intermediates produced by these reactions. Finally, bioaffinity HPLC is being evaluated as a separatory and analytical tool. The latter includes its use to characterize the noncovalent peptide—protein and protein—protein interactions which occur among the molecular forms of the neurophysin/hormone pathways. These experiments typify the impact of HPLC for both analytical and preparative separations in studies of biologically active peptides and proteins. 相似文献
3.
Kazeto Y Kohara M Miura T Miura C Yamaguchi S Trant JM Adachi S Yamauchi K 《Biology of reproduction》2008,79(5):938-946
Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated. 相似文献
4.
The polypeptide growth factor, multiplication stimulating activity (MSA), stimulates ornithine decarboxylase (ODC) activity in isolated porcine granulosa cells maintained under chemically defined conditions in vitro. The stimulatory action of MSA is saturable, and dose-dependent (0.1-1000 ng/ml). MSA effects are additive to those of luteinizing hormone (LH), but not those of follicle stimulating hormone (FSH). Stimulation of ODC activity by MSA requires cellular protein and RNA synthesis, and appears to be mediated independently of cyclic AMP. These observations provide the first demonstration of MSA action in the mammalian ovary. 相似文献
5.
6.
K Vuori R Myllyl? T Pihlajaniemi K I Kivirikko 《The Journal of biological chemistry》1992,267(11):7211-7214
Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another. 相似文献
7.
Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies. 总被引:3,自引:3,他引:0
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1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays 相似文献
8.
Y Baba A Arimura A V Schally 《Biochemical and biophysical research communications》1971,45(2):483-487
The effect of 2-hydroxy-5-nitrobenzyl bromide on the biological activity of a preparation of pure porcine LH and FSH-releasing hormone (LH-RH/FSH-RH) was reinvestigated. Since this treatment as well as performic acid and incubation with anhydrous trifluoroacetic acid, caused a complete inactivation of LH-RH/FSH-RH, tryptophan residue is thought to be essential for the biological activity of this polypeptide. 相似文献
9.
Biological activity and structural stability of N-deglycosylated recombinant human erythropoietin 总被引:1,自引:0,他引:1
A J Sytkowski L Feldman D J Zurbuch 《Biochemical and biophysical research communications》1991,176(2):698-704
Controversy exists regarding the functional role of N-linked oligosaccharides in the hormone erythropoietin. We have now examined the role of carbohydrates in the hormone's action using quantitative enzymatic deglycosylation. N-deglycosylated hormone exhibited full biological activity and potency in vitro. Denaturing with 6M urea and renaturing revealed that both the native and N-deglycosylated forms recovered full activity as long as the intrachain disulfide bonds remained intact. Therefore, receptor recognition, subsequent biological activity and maintenance of tertiary structure are intrinsic properties of the polypeptide chain of erythropoietin. 相似文献
10.
T L Low G B Thurman M McAdoo J McClure J L Rossio P H Naylor A L Goldstein 《The Journal of biological chemistry》1979,254(3):981-986
A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5. 相似文献
11.
E C Griffiths 《Hormone research》1976,7(3):179-191
With the structural characterization of the hypothalamic hormones, luteinizing hormone-releasing hormone (LH-RH), thyrotrophin-releasing (TRH), melanocyte-stimulating hormone release-inhibiting hormine (MIH), and growth hormone release-inhibiting hormone, (GH-RIH or somatostatin), it has been possible to investigate their enzymic inactivation by peptidases which are present at various sites in the body. Enzymes may play an important part in the control of polypeptide hormone levels and the peptidases acting on these four hypothalamic hormones may regulate the amount of TRH, LH-RH, MIH and somatostatin released from the hypothalamus, or their action at the level of the pituitary and their removal from the circulation. By studying the peptidase enzymes, further information may be obtained on the physiological mechanisms controlling the secretion and actions of hypothalamic hormones, as well as on the design of analogues with increased or competitive activity. 相似文献
12.
《Cell cycle (Georgetown, Tex.)》2013,12(12):1877-1882
Constitutively expressed cyclooxygenase-2 (COX-2) is a marker of tumor cell aggressiveness. Inducible COX-2 has also been described in cancer cells and localizes in the cancer cell nucleus, where formation of a complex of mitogen-activated protein kinase (MAPK) and COX-2 is antecedent to p53-dependent apoptosis. The stilbene resveratrol is a model pharmacologic activator of this pro-apoptotic mechanism. Physiological concentrations of thyroid hormone are anti-apoptotic in several types of tumor cells. A mechanism by which the hormone is anti-apoptotic is disruption of the nuclear MAPK-COX-2 complex. We review here the apoptosis-relevant effects of resveratrol and thyroid hormone and then speculate about the significance of convergence of these actions in cancer cells in the intact organism. Clinical activity of resveratrol may be modulated by normal tissue levels of endogenous thyroid hormone, and hypothyroidism in the cancer patient—whether spontaneous or induced by chemotherapeutic agents—may permit full expression of the apoptotic activity of the administered stilbene. Chronic pharmacologic inhibition of COX-2 may oppose the pro-apoptotic effect of resveratrol. 相似文献
13.
Immunoreactive Parathyroid Hormone in Circulation of Man 总被引:9,自引:0,他引:9
JOEL F. HABENER GINO V. SEGRE DAVID POWELL TIMOTHY M. MURRAY JOHN T. POTTSJUN. 《Nature: New biology》1972,238(83):152-154
WE have reported that parathyroid hormone (PTH) is secreted from the parathyroid in vivo as a polypeptide of eighty-four amino-acids, identical to the hormone stored in the glands (molecular weight of 9,500), but that the hormonal polypeptide is cleaved after it enters the general circulation1. A large hormonal fragment from this cleavage, with a molecular weight of approximately 7,500, has been identified in the circulation. The fragment differs immunologically from the hormone secreted and extracted from the glands1. To analyse the biological significance of the metabolism of the hormone and the chemical nature and hormonal activity of the large circulating fragment, we have developed radioimmunoassays that specifically measure the amino-terminal (N-assay) and carboxyl-terminal (C-assay) regions of the hormonal molecule. We now report that much higher concentrations of immunoreactive hormone are found in the general circulation by the C-assay than by the N-assay. The studies with the N-assay indicate that the large fragment has lost a portion of the amino-terminal sequence required for biological activity9. Since the fragment is present in much higher concentration than native uncleaved hormone, we must conclude that much of the immunoreactive PTH detected in the circulation is biologically inactive. 相似文献
14.
《Progress in growth factor research》1990,2(3):169-179
Anti-Müllerian hormone (AMH) is a dimeric glycoprotein member of the TGF-β family. It is synthesized by immature Sertoli cells, and, to a lesser degree, by adult Sertoli and granulosa cells. AMH is responsible for the regression of Müllerian ducts in the male fetus; it also has deleterious effects on the female fetal reproductive tract, destroying Müllerian primordia and germ cells, and masculinizing the fetal ovary on the rare occasions female fetuses become exposed to its effects. All other suggested actions for AMH—retardation of oocyte meiosis, inhibition of EGF receptor autophosphorylation, anti-cancer activity—have been reported with crude hormone preparations, and have not been confirmed using pure AMH. Its relatively limited sphere of action—the fetal genital tract—and the fact that it is secreted into the general circulation and can act at long range, imply that AMH is more like a hormone than a growth factor, but the complex interaction between hormones and growth factors make a formal distinction impossible. 相似文献
15.
Stephen M Cohen 《The EMBO journal》2014,33(9):937-938
Long non‐coding RNAs have become the focus of considerable interest over the past few years. Intriguing novel functions have been reported for lincRNAs. Three recent papers identify lincRNAs that work in a more conventional way—encoding protein—in each case a small polypeptide with an interesting biological activity (Magny et al, 2013 ; Pauli et al, 2014 ), (Bazzini et al, 2014 ). 相似文献
16.
T.J. McDonald M.A. Ghatei S.R. Bloom N.S. Track J. Radziuk J. Dupre V. Mutt 《Regulatory peptides》1981,2(5):293-304
The effect on plasma gastroenteropancreatic hormone levels on infusing the porcine gastrin-releasing peptide and bombesin into dogs demonstrated no qualitative difference in the spectrum of activity of the two peptides. Sustained elevations in plasma immunoreactive gastrin, pancreatic polypeptide, enteroglucagon, gastric inhibitory polypeptide, pancreatic glucagon and transient elevations in plasma insulin were seen during infusions of both peptides. The similar spectrum of activities and the structural homology between the two peptides suggests that the porcine gastrin releasing peptide is the porcine counterpart of the amphibian peptide bombesin. 相似文献
17.
P Crine N G Seidah F Gossard M Lis M Chrétien 《Revue canadienne de biologie / éditée par l'Université de Montréal》1980,39(1):35-53
The biosynthesis of numerous polypeptide hormones implicates two types of precursors: pre- and prohormones. The structure, characteristics, and role of these hormone precursors is discussed taking as examples parathyroid hormone and insulin. After this general introduction, the case of the common precursor for adrenocorticotropin and beta-lipotropin is then discussed. Its maturation proceeds through a serie of proteolytic steps which lead to the formation of various end products characterized each by a biological activity of its own. 相似文献
18.
An extract of porcine brain acetone powder incubated with thyrotropin-releasing hormone (TRH; pGlu-His-ProNH2) produces acid TRH (pGlu-His-Pro), histidine, and prolineamide. Fractionation of the brain extract by DEAE-cellulose chromatography produces three protein fractions which metabolize TRH. The activity of these fractions was characterized using TRH with a 3H-label on the histidine or proline as well as [His-3H]His-ProNH2. Fraction I contains pyroglutamate aminopeptidase and Fraction II contains TRH deamidase. Fraction III was found to contain a previously unrecognized enzyme which cleaves His-ProNH2 to histidine and proline. The histidylprolineamide imidopeptidase has been characterized. A competition study using a variety of compounds containing histidine or proline suggests that the best substrates for the imidopeptidase contain a free alpha-amino group on histidine and a blocked carboxyl group on proline, as is found in His-ProNH2. A survey of a variety of polypeptide hormones indicates that many of them inhibit the imidopeptidase activity. A kinetic study of the inhibition of the enzyme by adrenocorticotropic hormone (1-24) shows that the inhibition by polypeptide hormones is noncompetitive. We hypothesize that pituitary hormones may stimulate the production of (cyclo)-His-Pro by inhibiting alternate routes of TRH metabolism. 相似文献
19.
The polypeptide hormones insulin, glucagon, thyrotropin (TSH), pregnant mare serum gonadotropin (PMSG) and adrenocorticotropin (ACTH) stimulated the growth of the Tetrahymena, and the non-hormone polypeptides (bovine serum albumin (BSA), protamine) had a similar effect. Re-exposure after 24 h accounted for a greater growth stimulation than pre-exposure alone in cultures treated with TSH and PMSG, and re-exposure after 7 days had such effect in all polypeptide-treated cultures. It follows that the non-hormone polypeptides had a similar imprinting potential to the polypeptide hormone. The non-hormone polypeptides were also able to cross-imprint for one another, i.e. pre-exposure to one enhanced the binding capacity of the cells for the other on re-exposure, and vice versa. A single treatment with a polypeptide hormone or a non-hormone polypeptide did in itself stimulate the growth of the Tetrahymena for as long as 1 week. 相似文献
20.
R J Wojcikiewicz P A Kent J N Fain 《Biochemical and biophysical research communications》1986,138(3):1383-1389
Thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) and acetylcholine stimulated high affinity GTPase activity in GH3 cell membrane preparations. The effects of acetylcholine and VIP were blocked by pretreatment of cultured cells with pertussis toxin and cholera toxin respectively. Such pretreatment, which causes covalent modification of the guanine nucleotide-binding proteins (G-proteins) of adenylate cyclase, did not, however, block the effects of TRH on GTPase activity or phosphoinositide breakdown. These data suggest that TRH receptors interact with a G-protein discrete from those associated with regulation of adenylate cyclase activity. 相似文献