Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of mycobacterial growth inhibitory factor (MycoIF) to trypsin, chymotrypsin, or neuraminidase decrease its ability to produce intracellular inhibition of mycobacterial growth within macrophages, suggesting that MycoIF was a glycoprotein. MycoIF was unaffected by deoxyribonuclease or ribonuclease. Supernatant fluids from antigenically stimulated H37Ra-immunized mouse spleen cells exposed to puromycin were unable to produce significant intracellular inhibition. This indicated that the presence of MycoIF activity in supernatant fluids required protein synthesis. The filtration of MycoIF-containing supernatant fluids on Sephadex G-150 demonstrated that significant MycoIF activity appeared only in those fractions which eluted on the downward side of the serum albumin peak. Based on protein standards filtered through the Sephadex gel, the molecular weight of MycoIF was calculated to be between 20,000 and 35,000. These calculations assumed that MycoIF is a globular protein. Attempts to purify MycoIF by anion exchange chromatography (diethylaminoethylcellulose) was not successful.
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PMID:Molecular weight and other characteristics of mycobacterial growth inhibitory factor produced by spleen cells obtained from mice immunized with viable attenuated mycobacterial cells. 81 60

Mycobacterial growth inhibitory factor (MycoIF), which inhibits the intracellular multiplication of virulent tubercle bacilli within normal peritoneal macrophages in vitro, was tested for its ability to inhibit the migration of normal peritoneal exudate cells. The migration of peritoneal exudate cells was not inhibited by MycoIF. It was also shown that normal peritoneal macrophages infected with virulent Mycobacterium tuberculosis, strain H37Rv, required 72 h of incubation with spleen cell culture supernatant fluids containing MycoIF in order to inhibit intracellular bacillary multiplication. Treatment of infected macrophages with trypsin before their exposure to MycoIF abolished the ability of MycoIF to inhibit intracellular mutiplication of tubercle bacilli. Incubation of infected macrophages with goat anti-mouse globulin before their exposure to MycoIF also blocked the action of MycoIF.
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PMID:Macrophage migration inhibitory activity of mycobacterial growth inhibitory factor and the effect of a number of factors on mycobacterial growth inhibitory factor activity. 81 61

The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.
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PMID:The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells. 117 Aug 80

Cultures of the human mammary carcinoma line ZR-75-1 secrete a growth inhibitory factor (GIF) that, when diluted, slows the growth of MDA-MB-231 and MCF-7 cells. Undiluted "conditioned" media prevents cell division from occurring in both human breast cancer lines. ZR-75-1 cells are unaffected by this factor. The amount of GIF in the culture media is related to the confluency of the ZR-75-1 cells. The activity of this GIF is not altered by DNAse or RNAse but is destroyed by heating or trypsin. Growth inhibition is 85-90% reversible if conditioned media is replaced with fresh media.
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PMID:Secretion of a growth inhibitory factor by ZR-75-1 human breast cancer cells. 342 94

Certain properties of experimental pellicles formed by the adsorption of salivary components on hydroxyapatite surfaces change over time. To determine whether enzymes likely to be present in the oral environment could induce such changes, pellicles were treated with saliva which had been incubated for 18 h at 35 degrees C to promote the elaboration of microbial enzymes. This treatment markedly reduced the numbers of Streptococcus mutans MT3 and JBP and S. sanguis FC-1 and C5 cells which attached, but it had little or no effect on the attachment of S. mitis RE7, Actinomyces viscosus LY7 and CK-8, Bacteroides gingivalis 381, or B. melaninogenicus subsp. intermedius 581. Heating the incubated saliva at 60 degrees C for 30 min partially reduced its pellicle-modifying activity, whereas heating at 80 degrees C for 30 min or 100 degrees C for 15 min completely eliminated such activity. This indicated that the saliva contained heat-labile substances, presumably enzymes, which could affect the pellicle receptors involved in the attachment of S. mutans and S. sanguis. Treatment of saliva-treated hydroxyapatite with commercially obtained enzyme preparations also affected bacterial attachment. Thus, treatment with galactose oxidase reduced the numbers of the S. mutans strains which attached, whereas treatment with neuraminidase reduced the adsorption of S. sanguis FC-1 but not that of S. sanguis C5. Treatment with beta-glucosidase preparations derived from almonds significantly reduced the attachment of all of the streptococcal strains studied, but, when subjected to isoelectric fractionation, the adherence-inhibiting activity did not correlate directly with beta-glucosidase activity. Treatment of the pellicles with trypsin or eight other glycosidases did not affect streptococcal attachment. Exposure of the enzymatically modified pellicles to fresh saliva did not restore the streptococcal receptors. Collectively, the data suggest that some bacterial receptors in the pellicle coating of teeth can be modified by enzymes likely to be present in the oral environment, and these interactions may affect oral bacterial ecology.
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PMID:Enzymatic modification of bacterial receptors on saliva-treated hydroxyapatite surfaces. 628 Nov 93

Human peripheral blood mononuclear leucocytes (PBML) stimulated with concanavalin A (Con A) or phytohaemagglutinin (PHA) produced a soluble factor which inhibits lung fibroblast DNA synthesis and growth. Lymphocyte enriched preparations produced significant growth inhibitory activity in the presence of PHA whereas media from adherent mononuclear cells incubated in the presence of the mitogen did not contain similar activity. This fibroblast growth inhibitory factor (FGIF) was non-dialysable, heat stable and resistant to pH 5. FGIF was also resistant to treatment with chymotrypsin and phosphodiesterase but partially sensitive to treatment with trypsin. Interestingly, there was significant suppression of FGIF production by PBML cultured with PHA in the presence of low concentrations of chrysotile asbestos (5-25 micrograms/ml). In this regard, asbestos (25 micrograms/ml) was not cytotoxic for lymphocytes but had a damaging effect on monocytes as evidenced by the release of lactate dehydrogenase (LDH) a cytoplasmic enzyme, in their culture media. These findings indicate that stimulated lymphocytes have the ability to inhibit fibroblast proliferation by releasing FGIF and that asbestos interfere with this process. Thus, while FGIF may regulate the extent of connective tissue proliferation during normal repair process, suppression of its production by asbestos may contribute to excessive fibroblast accumulation and fibrosis.
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PMID:In vitro suppression of fibroblast growth inhibitory lymphokine production by asbestos. 687 28

To determine its active site, growth inhibitory factor (GIF), a central nervous system-specific metallothionein-like protein, was digested with trypsin followed by Staphylococcus aureus protease V8 digestion. Of 5 peptide fragments separated from trypsin-digested GIF by reverse-phase high pressure liquid chromatography and gel filtration, only GIF1-26 or longer peptides showed growth inhibitory activity on cortical neurons in culture. A shorter peptide, GIF5-23, which was obtained by further digestion of GIF1-26 with V8 protease, also showed growth inhibitory activity. However, a synthetic peptide corresponding to GIF5-23 did not show growth inhibitory activity. Metal-free GIF1-26 prepared by acid treatment showed a similar level of growth inhibitory activity to that of metal-containing GIF1-26, indicating that metal in the peptide does not affect the activity. Treatment of metal-free GIF1-26 with beta-mercaptoethanol resulted in the loss of activity. The CD spectrum of beta-mercaptoethanol-treated metal-free GIF1-26 was different from that of nontreated metal-free GIF1-26. These results indicate that the N-terminal portion of GIF is required for growth inhibitory activity and that folding of the peptide via S-metal bonding is critical for biological activity.
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PMID:The N-terminal portion of growth inhibitory factor is sufficient for biological activity. 785 23

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.
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PMID:Identification of mouse brain proteins associated with isoform 3 of metallothionein. 1580 40

Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.
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PMID:Metallothionein-3 is a component of a multiprotein complex in the mouse brain. 1701 72

Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn(II) and Cu(I) ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn(II)-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn(II) competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn(II) release processes. The Cu(I)-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn(II) release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
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PMID:Metal ion release from metallothioneins: proteolysis as an alternative to oxidation. 2383 14


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