Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P05231 (interleukin-6)
 23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.
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PMID:Inactivation of interleukin-6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl-transferase activities. 835 88

Obtained from pSj5, the cDNA gene encoding GST antigen of Schistosoma japonicum (Philippine strain) was ligated with efficient temperature-dependent PBV220 vector which was constructed in CAPM, and then introduced into host bacterium-DH5 alpha (E. coli) by transformation. Transformants were selected by ampicillin and recombinant clones were identified by restriction mapping. The result showed that recombinant clone 43 was the one carrying recombinant plasmid PBV 220 with the correct insertion of the gene fragment. The GST expression ability of clone 43 was investigated by GST enzymic activity assay and SDS-PAGE. A relatively high level of GST enzymic activity was expressed by this clone under the temperature-dependent condition, that is, cultured at 30 degrees C and expressed at 42 degrees C. A more strongly stained 26 kDa protein band was identified by SDS-PAGE. The result indicated that GST of S. japonicum (Philippine strain) could be expressed not only by IPTG induction, but also by the temperature-dependent method.
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PMID:The temperature--dependent expression of GST of Schistosoma japonicum (Philippine strain). 836 8

The recombinant soluble human interleukin-6 receptor (srhIL-6R) was expressed in Escherichia coli as a non-glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL-6R could be renaturated by an in vitro folding procedure using L-arginine and the glutathione-redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL-6-Sepharose column. The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145-345) of the human interleukin-6 receptor (IL-6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL-6 binding, it could be used to establish a cell-free IL-6-binding assay, In this assay, the srhIL-6R bound IL-6 with an affinity of Kd = 1.5 nM as measured by Scatchard-plot analysis. When 125I-IL-6 was chemically cross-linked to the purified srhIL-6R and analyzed by SDS/PAGE, several 125I-IL-6-containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL-6/IL-6R complex. The srhIL-6R was shown to exhibit biological activity, i.e. it stimulated acute-phase protein synthesis in the recently established human hepatoma cell line HepG2-IL-6 which does not express the IL-6-binding subunit of the IL-6R complex on the cell surface.
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PMID:Recombinant soluble human interleukin-6 receptor. Expression in Escherichia coli, renaturation and purification. 836 10

Affinity cross-linking of membrane bound 125I-interleukin-6 (IL-6) on several cell lines revealed a three-band pattern of IL-6-containing cross-linked complexes with molecular masses of 100, 120, and 150 kDa. To identify the membrane components that were associated with IL-6 in the three complexes, we employed the Denny-Jaffe reagent, a heterobifunctional, cleavable cross-linker that allows the transfer of 125I from the ligand to its receptor. Samples cross-linked with Denny-Jaffe reagent were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis in which the cross-linker was cleaved prior to the second dimension. This analysis revealed that IL-6 directly associates with a 130-kDa membrane protein thus allowing the formation of the 150-kDa complex. In addition, both the 100- and 120-kDa cross-linked complexes were shown to include an 80-kDa membrane glycoprotein associated with one and two IL-6 molecules, respectively.
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PMID:Direct association of interleukin-6 with a 130-kDa component of the interleukin-6 receptor system. 842 Sep 83

The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l.mol-1.cm-1 was calculated from the ultraviolet spectrum of the soluble IL-6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human 125I-labeled IL-6 with low affinity (Kd = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble 125I-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.
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PMID:Soluble human interleukin-6 receptor. Expression in insect cells, purification and characterization. 853 17

Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin-labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins.
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PMID:Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family. 863 35

The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein 1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.
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PMID:Purification and characterization of the soluble interleukin-6 receptor from human plasma and identification of an isoform generated through alternative splicing. 866 2

The small dermatan sulfate proteoglycans decorin and biglycan are efficiently internalized by a variety of cells of mesenchymal origin. This process is modulated, at least under tissue culture conditions, by cell surface-associated heparan sulfate proteoglycans. Receptor proteins of 51 and 26 kDa, respectively, bind to leucine-rich repeat structures of the core proteins of the small proteoglycans but also to highly sulfated domains of heparan sulfate. The 51 kDa protein was purified from rat brain tissue by subcellular fractionation, heparin affinity chromatography and subsequent SDS-PAGE, and was used for raising a polyclonal antiserum. Affinity-purified antibodies also recognize the 26 kDa protein and a few other low molecular weight proteins, suggesting that these proteins represent proteolytic degradation products of the 51 kDa receptor. By confocal laser microscopy, it could be demonstrated that the affinity-purified antibody reacted at 0 degree C with a protein that became internalized and was transported to a perinuclear compartment during 15 min of incubation at 37 degrees C. These findings provide direct evidence that the receptor protein(s) are internalized together with the ligand and reach an endosomal compartment where further sorting can occur.
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PMID:Isolation and cellular localization of the decorin endocytosis receptor. 898 Sep 2

The effect of eight different Chinese medicinal herbs (CMHs) on lymphocytes was studied in vitro using murine spleen cells. Among the studied eight CMHs, Astragalus membranaceus and Oldenlandia diffusa markedly stimulated murine spleen cells to proliferate. The responder cells for CMHs were B cells, because the response was depleted by the treatment of spleen cells with anti-immunoglobulin (i.g.) antibody and complement and after purification by nylon wool column. This response was not due to contamination by lipopolysaccharide (LPS), because CMHs could stimulate C3H/HeJ spleen cells which are low responders to LPS. CMHs enhanced the production of Ig. CMHs also enhanced the induction of allo-antigen specific cytotoxic T lymphocytes. However, CMHs had no effect on natural killer cells. Furthermore, CMHs stimulated macrophages to produce interleukin-6 and tumor necrosis factor. The electroelution of the proteins from SDS-PAGE gel showed that the active components of Oldenlandia diffusa had an apparent molecular weight of 90-200 kD and were sensitive to pronase E and NaIO4 treatment, suggesting glycoproteins in nature. These results suggest that CMHs have immunomodulating activity in vitro and this activity could be used clinically for the modulation of immune responses.
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PMID:Immunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular. 956 40

Uteroglobin (UG) is a steroid-inducible, multifunctional, secreted protein with antiinflammatory and antichemotactic properties. Recently, we have reported a high affinity UG-binding protein (putative receptor), on several cell types, with an apparent molecular mass of 190 kDa (Kundu, G. C., Mantile, G., Miele, L., Cordella-Miele, E., and Mukherjee, A. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2915-2919). Since UG is a homodimer in which the 70 amino acid subunits are connected by two disulfide bonds, we sought to determine whether UG monomers also interact with the 190-kDa UG-binding protein and if so, whether it has the same biological activity as the dimer. Surprisingly, we discovered that in addition to the 190-kDa species, another protein, with an apparent molecular mass of 49 kDa, binds reduced UG with high affinity and specificity. Both 49- and 190-kDa proteins are readily detectable on nontransformed NIH 3T3 and some murine cancer cells (e. g. mastocytoma, sarcoma, and lymphoma), while lacking on others (e.g. fibrosarcoma). Most interestingly, pretreatment of the cells, which express the binding proteins, with reduced UG dramatically suppresses extracellular matrix (ECM) invasion, when such treatment had no effect on fibrosarcoma cells that lack the UG-binding proteins. Tissue-specific expression studies confirmed that while both 190- and 49-kDa UG-binding proteins are present in bovine heart, spleen, and the liver, only the 190-kDa protein is detectable in the trachea and in the lung. Neither the 190-kDa nor the 49-kDa protein was detectable in the aorta. Purification of these binding proteins from bovine spleen by UG-affinity chromatography and analysis by SDS-polyacrylamide gel electrophoresis followed by silver staining identified two protein bands with apparent molecular masses of 40 and 180 kDa, respectively. Treatment of the NIH 3T3 cells with specific cytokines (i.e. interleukin-6) and other agonists (i.e. lipopolysaccharide) caused a substantially increased level of 125I-UG binding but the same cells, when treated with platelet-derived growth factor, tumor necrosis factor-alpha, interferon-gamma, and phorbol 12-myristate 13-acetate, did not alter the UG binding. Taken together, these findings raise the possibility that UG, through its binding proteins, plays critical roles in the regulation of cellular motility and ECM invasion.
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PMID:Uteroglobin (UG) suppresses extracellular matrix invasion by normal and cancer cells that express the high affinity UG-binding proteins. 971 16


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