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Query: UNIPROT:P06889 (Mol)
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The domain structures and stabilities of fragments isolated from the so-called 'hep 2' region of plasma fibronectin have been investigated by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The 30 kDa hep-2A fragment contains three type III modules (III12 to III14), whereas the 40 kDa hep-2B fragment contains four such modules (III12 to III15). Melting of these fragments at neutral pH was irreversible and accompanied by rapid aggregation. In contrast, melting was completely reversible in 50 mM-glycine at pH 2.7, where DSC measurements revealed the presence of three independently folded domains in 30kDa hep-2A and four in 40 kDa hep-2B. That each domain represented a single module was confirmed by measurements with four single-module subfragments, all of which melted reversibly, even at neutral pH. At neutral pH in the presence of 6 M-urea, 30 kDa hep-2A melted reversibly in a sharp peak from which only two transitions could be resolved by deconvolution. Only the larger of these was stabilized by heparin and was assigned to modules III13 and III14. Upon isolation, module III13 melted at lower temperature than in the parent fragment where it is stabilized through an interaction with module III14. We conclude that all type III modules in the hep-2 region of fibronectin constitute independently folded domains. Modules III13 and III14 form a highly co-operative structure through functionally significant interactions that can be disrupted with acid or sufficient concentrations of urea or guanidinium chloride.
J Mol Biol 1992 Oct 20
PMID:Domain structure and domain-domain interactions in the carboxy-terminal heparin binding region of fibronectin. 143 92

Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].
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PMID:Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin. 144 73

Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.
J Mol Biol 1992 Nov 20
PMID:Folding on the ribosome of Escherichia coli tryptophan synthase beta subunit nascent chains probed with a conformation-dependent monoclonal antibody. 145 47

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.
Mol Reprod Dev 1992 Dec
PMID:Immunological evidence for a P2 protamine precursor in mature rat sperm. 147 78

Charcoal-dextran stripped serum/plasma supplemented media specifically inhibit the proliferation of estrogen-sensitive cells in culture conditions; estrogens cancel this effect. Here, we further characterize this phenomenon using human estrogen-sensitive breast cancer MCF7 cells and human serum/plasma. The serum/plasma-borne inhibitory activity (estrocolyone-I) is a non-dialyzable, heat-stable (60 degrees C x 2 h), protease-sensitive macromolecule and it is not extractable by organic solvents. Estrocolyone-I activity is retained after dialysis against 6 M urea or 10-100 mM dithiothreitol; however, simultaneous treatment with 6 M urea and 10-100 mM dithiothreitol completely abolishes its inhibitory activity. The inhibitory effect of serum is not due to serum albumin, nor to estrogen trapping by albumin or by sex hormone-binding globulin. Substantial purification was achieved by a combination of chromatographic techniques (dye-affinity, ion exchange, hydrophobic interaction chromatography). Estrocolyone-I activity seems to be due to a protein of an apparent native Mw of 70-80 kDa and an isoelectric point of 4.5-4.8.
J Steroid Biochem Mol Biol 1992 Dec
PMID:A plasma-borne specific inhibitor of the proliferation of human estrogen-sensitive breast tumor cells (estrocolyone-I). 147 62

Based on the molecular theory of protein structure the de novo protein was designed in order to obtain the tertiary fold which has not yet been observed in natural proteins, namely four-stranded antiparallel beta-sheet covered by two alpha-helixes. The gene coding for this protein (named albebetin) was chemically synthesized, cloned in plasmid with SP6 phage promoter and expressed in mRNA-dependent cell-free translation system. An approach was developed to study albebetin using only nanogram amounts of radio labelled protein without previous purification. The preliminary analysis of its structure by gel-filtration, urea-gradient electrophoresis and limited proteolysis revealed compactness and stability of the de novo protein.
Mol Biol (Mosk)
PMID:[De novo proteins with a given spatial structure: new approaches to design and analysis]. 149 70

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.
Mol Biol (Mosk)
PMID:[Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]. 150 64

In GH4C1 rat pituitary cells, cell swelling stimulates prolactin (PRL) secretion by increasing Ca2+ influx through nifedipine-sensitive Ca2+ channels; however, the mechanism by which cell swelling opens Ca2+ channels is still unclear. To evaluate the role of protein kinase C (PKC) in this phenomenon, we studied the effect of down-regulating PKC by 12-h pretreatment with phorbol ester or by treatment with H-7, a protein kinase C inhibitor. Cell swelling induced by either 27% medium hyposmolarity or 80 mM isotonic urea caused a prompt rise in both [Ca2+]i and PRL secretion in otherwise untreated control GH4C1 cells. Removal of medium Ca2+ enhanced the osmotically induced cell swelling but prevented the increase in [Ca2+]i and PRL secretion. Both PKC down-regulation and H-7 suppressed the cell swelling-induced increases in [Ca2+]i concentration and PRL secretion, although they enhanced the induced cell volume expansion. Our data indicate that in GH4C1 cells PKC plays an important positive modulating role in the osmotic opening of plasmalemma Ca2+ channels, a critical component of the early transduction chain by which cell swelling causes PRL secretion in tumor-derived clonal pituitary cells.
Mol Cell Endocrinol 1992 Aug
PMID:Protein kinase C modulates cell swelling-induced Ca2+ influx and prolactin secretion in GH4C1 cells. 151 83

Graves' disease is an autoimmune thyroid disease characterized by the presence of pathogenic autoantibodies to the TSH receptor (TSH-R). By using polymerase chain reaction, the extracellular region of the human TSH-R cDNA has been amplified and used to prepare recombinant TSH-R (extracellular) protein fused with glutathione-S-transferase (GST). Purification of the recombinant TSH-R (extracellular)-GST fusion protein was achieved by preparative gel electrophoresis in SDS or by preparative isoelectric focusing in urea. Following removal of SDS by detergent exchange or urea by dialysis, the purified recombinant receptor preparations were assessed for binding to the hormone or to autoantibodies from Graves' disease patients. The purified recombinant receptor preparations fail to show any binding to the hormone or autoantibodies either by inhibition of binding assays or by immunoblotting. The results imply that the correct folding and/or post-translational modifications of the polypeptide chain which are not achieved in recombinant proteins produced in Escherichia coli may be important for the binding of the hormone or Graves' disease autoantibodies to the TSH-R. The recombinant receptor prepared in this manner will be useful for immunological and cellular investigations in patients with Graves' disease.
J Mol Endocrinol 1992 Apr
PMID:Expression of a human thyrotrophin receptor fragment in Escherichia coli and its interaction with the hormone and autoantibodies from patients with Graves' disease. 151 18

The interaction between hen lysozyme and urea has been investigated using 1H nuclear magnetic resonance spectroscopy. Chemical shift changes for resonances of a number of residues in the vicinity of the active site of the protein have been observed in the presence of urea prior to denaturation. These shifts are similar to those induced in the hen lysozyme spectrum by the specific binding of N-acetylglucosamine (GlcNAc) in site C of the active site cleft, indicating that urea and GlcNAc induce a similar conformational change in the enzyme. This implies that the conformational changes experienced by the enzyme on the binding of GlcNAc oligosaccharides are the consequence of interactions, possibly hydrogen bonding, involving the N-acetyl group of the sugar residue bound in site C, rather than the result of contacts between the protein and the pyranose rings of the oligosaccharides. This suggests that hen lysozyme employs an induced fit type mechanism to discriminate for N-acetylated saccharides as substrates.
J Mol Biol 1992 Sep 05
PMID:1H nuclear magnetic resonance studies of the interaction of urea with hen lysozyme. Origins of the conformational change induced in hen lysozyme by N-acetylglucosamine oligosaccharides. 152 4


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