A radioimmunoassay for parathymosin

Panneerselvam, C.; Caldarella, Janet; Horecker, B.L.

doi:10.1016/0022-1759(87)90496-0

Cited by 18 publications

(16 citation statements)

References 11 publications

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“…Peptide fragment thymosin t4 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and the analog [Tyr12]-thymosin t4 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were prepared by solid phase synthesis on an automatic Synostat-peptide synthesizer (Biotronik, Maintal, Germany) using 4-(hydroxymethyl)-phenoxymethyl-copoly-(styrene-1% divinyl-benzene)-resin preloaded with FMOC-Ser (tBu)-OH (for each synthesis 0.33 g = 0.26 mmol). The synthetic analog [TyrlZ]-thymosin /34 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) was radioiodinated by the chloramine T method as previously reported for tracers prepared for e-thymosin radioimmunoassays 15'16. All amino acids were coupled twice in DMF in threefold excess in the presence of 0.35 g BOP/0.11 g HOBt and 1.6 ml diisopropylethylamine (25% in DMF).…”

Section: Methodsmentioning

confidence: 99%

Evidence for the extranuclear localization of thymosins in thymus

et al. 1992

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A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.

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Section: Methodsmentioning

confidence: 99%

Evidence for the extranuclear localization of thymosins in thymus

et al. 1992

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“…After purifying the protein to homogeneity and analyzing its primary structure by peptide sequencing, it became evident that this nuclear protein is identical to prothymosin a (ProTa) [21* A group of peptides, named thymosins, were originally isolated from calf thymus tissue, particularly from calf thymosin fraction 5 [3,4], but have since been shown to be widely distributed in mammalian cells and tissues [5][6][7]. Thymosin ty, the first peptide to be isolated from thymosin fraction 5, has been shown to be the N-terminal fragment (amino acid residues l-28) of a larger polypeptide designated as prothymosin cx (111 amino acid residues) 121.…”

Section: Introductionmentioning

confidence: 99%

Identification of a low‐M_r acidic nuclear protein as prothymosin α

Palvimo¹,

Linnala-Kankkunen²

1990

FEBS Letters

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We have purified to homogeneity a lij-kDa perchloric acid (PCA)-soluble protein from rat thymus nuclei. This highly acidic protein showed a M, of ca. 30 kDa in acetic acid/urea gels, probabty due to oligomer formation. Sequence analysis of internal tryptic and the~ol~c peptides revealed that the purified protein is, in fact, prothymosin u, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin G( is a rather abundant nuclear protein in rat thymus; its concentration is comparable to that of a well-characterized nonhistone protein HMG-14. The subcellular localization and physicochemical properties of prothymosin c( suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.

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“…One may speculate that many MTI‐II molecules, compared with GR molecules, are required to exert the inhibitory activity. Indeed, there is 105–326 µg (9.2–28.4 nmol) ZnBP per g of liver [12,17,33–35], and the concentration of GR is ≈ 30 pmol·g −1 liver [36]. Therefore, MTI‐II may inhibit the nuclear binding of GR in vivo .…”

Section: Discussionmentioning

confidence: 99%

Purification and primary structure of a macromolecular‐translocation inhibitor II of glucocorticoid‐receptor binding to nuclei from rat liver

Okamoto

Isohashi

2000

European Journal of Biochemistry

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The nuclear binding of the activated glucocorticoid-receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI-I, MTI-II and MTI-III). In this study, we purified the most potent of the three species (MTI-II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two-dimensional PAGE. Purified MTI-II inhibits GR binding to DNA containing glucocorticoid-response elements. To obtain the amino acid sequence of MTI-II, we digested the MTI-II with endopeptidases. The N-terminal amino acid sequences of the four digested fragments indicated that MTI-II is an 11.5-kDa Zn 2+ -binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI-II (ZnBP, parathymosin) and that GR binds to the MTI-II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.Keywords: glucocorticoid receptor; nuclear-translocation inhibitor; parathymosin; purification; zinc-binding protein.When nuclei or DNA were incubated with increasing concentrations of cytosol containing activated (transformed) glucocorticoid receptor (GR), receptor binding to nuclei or DNA reached apparent saturation [1±4]. Several laboratories have reported that this pseudo-saturation is due to the presence of an endogenous macromolecular inhibitor (or inhibitors) in the cytosol of rat liver [1±5] and rat hepatoma cells [6]. With regard to the physiological role of these inhibitors, it is speculated that the target cells may have an inherent mechanism that regulates the interaction of GR with the genomic machinery and thereby controls hormone responses at the cellular level [2,5]. Alternatively, the inhibitor may play a role in the release of the receptor from its nuclear-binding state [5]. We found that AH 130 ascites tumor cells (glucocorticoidresistant cells) have approximately five times more inhibitory activity than cells from rat liver [7], and that the inhibitory activity in regenerating rat liver following 70% hepatectomy is increased significantly during periods of DNA synthesis and subsequent mitosis and then decreases to normal level [8]. Thus, the inhibitors may be involved in cell proliferation by inhibiting GR binding to nuclei.Previously, we separated the macromolecular-translocation inhibitors (MTIs), which inhibit GR binding to nuclei in vitro, from the cytosol of rat liver [9] and other cells [7,8] into at least three components (MTI-I, MTI-II and MTI-III). Recently, we purified MTI-III to apparent homogeneity and characterized i...

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A radioimmunoassay for parathymosin

Cited by 18 publications

References 11 publications

Evidence for the extranuclear localization of thymosins in thymus

Evidence for the extranuclear localization of thymosins in thymus

Identification of a low‐M_r acidic nuclear protein as prothymosin α

Purification and primary structure of a macromolecular‐translocation inhibitor II of glucocorticoid‐receptor binding to nuclei from rat liver

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A radioimmunoassay for parathymosin

Cited by 18 publications

References 11 publications

Evidence for the extranuclear localization of thymosins in thymus

Evidence for the extranuclear localization of thymosins in thymus

Identification of a low‐Mr acidic nuclear protein as prothymosin α

Purification and primary structure of a macromolecular‐translocation inhibitor II of glucocorticoid‐receptor binding to nuclei from rat liver

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Identification of a low‐M_r acidic nuclear protein as prothymosin α