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)

A function of serum in the growth medium for primary monkey kidney cells has been shown to be inhibition of proteolytic enzymes. Serum inactivates the residual trypsin remaining from enzymatic digestion of the kidneys and the proteolytic enzymes subsequently synthesized by the cells. Freshly trypsinized cells could be grown to monolayers in the absence of serum provided that they were repeatedly washed to remove residual trypsin. In the absence of serum, cell growth ceased on the 4-5th day after initiation of the culture, at which time the culture fluids became active proteolytically. When the 5th day fluids were replaced with fresh serum-free medium, cell growth was accelerated and a monolayer was attained by the 7th day. If cells were grown in the absence of whole serum but in the presence of medium containing alpha globulins or fetuin which inhibit both trypsin and cell proteases, such cultures grew as well as cultures containing serum. The sterilization of trypsin for use in digestion of tissues and cell cultures poses a serious problem. After filtration through 0.22 micron filters, trypsin preparations may still contain adventitious viruses, mycoplasma and minute forms of pseudomonas and other bacteria or bacteria-produced toxins, which pass the membrane pores. A process of purifying and sterilizing trypsin without deleteriously affecting its proteolytic activity is described.
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PMID:Problems related to the use of serum and trypsin in the growth of monkey kidney cells. 2 71

Attachment of washed Mycoplasma gallisepticum cells to glass was quantified with organisms in which membrane lipids were labelled with 3H. Siliconization of the test tubes decreased attachment, while centrifugation increased it. Attachment increased with temperature, decreased with increasing pH and ionic strength of the attachment mixture, but was unaffected by Ca2+, Mg2+ and EDTA. This suggests that ionic bonds, but not salt bridges, participate in the attachment process. Glycophorin, the major receptor responsible for M. gallisepticum attachment to erythrocytes, partially inhibited the attachment of the organisms to glass. However, bovine serum albumin also decreased attachment. Extensive pretreatment of the organisms with trypsin decreased their ability to attach to glass by about 35 to 40%. Trypsin and pronase failed to detach the organisms already bound to glass, suggesting that external mycoplasma cell components, other than membrane proteins, also participate in attachment of the organisms to glass.
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PMID:Adherence of Mycoplasma gallisepticum to glass. 3 84

The human pathogen Mycoplasma pneumoniae adheres to a variety of cells, including erythrocytes. A hemadsorption technique was developed to quantitate adherence by photometric measurement of lysates of erythrocytes that attached to sheets of M. pneumoniae grown in cups of Linbro plates. Attachment of sheep erythrocytes (SE) increased with higher ionic strength, was unaffected by minor pH variations (6 to 9), and was blocked by anti-M. pneumoniae antiserum, but was not inhibited by a variety of sugars, amino acids, and bovine serum albumin. The reaction was time and temperature dependent. The temperature curve showed peaks at 14 and 28 degrees C with untreated SE but only one peak at about 38 degrees C with glutaraldehyde-treated SE. The temperature dependence indicated involvement of either metabolic or membrane activities in the binding process. Trypsin treatment of the M. pneumoniae sheet abolished adherence of SE but was only partially effective with human erythrocytes and noneffective with rabbit erythrocytes. The binding capacity of the mycoplasma cells for SE was restored by incubation in growth medium for 3 to 4 h; this restoration was inhibited by 10 mug of chloramphenicol per ml. Neuraminidase treatment of SE removed their attachment capacity but had no effect on attachment of rabbit erythrocytes and only a slight effect on attachment of human erythrocytes. Pretreatment of M. pneumoniae with neuraminic acid partially blocked the adherence of SE, whereas rabbit erythrocyte attachment was not affected. Attached SE could be detached by trypsin, but not by neuraminidase. For human and rabbit erythrocytes, the results suggest binding mechanisms other than the interaction between neuraminidase-sensitive receptors and protein-containing binding sites shown for SE.
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PMID:Adherence of erythrocytes to Mycoplasma pneumoniae. 3 34

The biochemical nature of the neuraminidase-sensitive Mycoplasma pneumoniae receptor site on human lung fibroblast cells was studied. Purified, mixed sialoglycolipid (ganglioside) preparations from human and bovine tissues did not bind to M. pneumoniae organisms and block their subsequent attachment to fibroblasts. Fibroblasts incubated for 24 h in sialoglycolipid solutions to increase the ganglioside content of their membranes did not show increased pathogen attachment when later incubated with mycoplasmas. HeLa cells grown in the presence of sodium butyrate to increase GM3 ganglioside levels likewise did not have significantly increased uptake of M. pneumoniae organisms. Treatment of fibroblasts with enzymes indicated that the mycoplasma receptor site is trypsin and papain resistant but Pronase sensitive. Pronase digests of fibroblast membranes contained a product(s) which combined with M. pneumoniae cellls and cosedimented with them during centrifugation. Glycoproteins, purified from fibroblast membranes by a lithium diiodosalicylate solubilization technique, similarly bound to M. pneumoniae organisms. Collectively, these data suggest that the major component of the M. pneumoniae receptor site is a sialoglycoprotein with little or no lipid.
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PMID:Interaction of Mycoplasma pneumoniae with human lung fibroblasts: role of receptor sites. 11 49

Rates of attachment and ingestion of Mycoplasma pulmonis by mouse peritoneal macrophages and the effect of the organism on phagocyte glucose metabolism were studied in vitro. Antimycoplasma antibody, but not complement, enhances attachment of mycoplasma to macrophages. Antibody-mediated attachment is not affected by the length of time the cell is maintained in culture or its adherence to a glass surface. Adherence of mycoplasma by nonimmunologic means to cell spread on glass is significantly greater than attachment to macrophages in suspension. Mycoplasmas attached to macrophage membrane do not increase glucose metabolism via the hexose monophosphate shunt. Because of this, the rate of glucose metabolism by macrophages is an accurate quantitative correlate of the recognition and ingestion stage of phagocytosis of mycoplasmas. Antibody induces a more rapid rate of particle ingestion than does altering the mycoplasma by proteolysis. Complement does not augment the rate of ingestion induced by antimycoplasma antibody. By analogy with enzyme and absorption kinetics, it appears likely that differences observed between phagocytosis induced by antibody and that induced by trypsin are due to different rates of recognition between particles rather than different ingestion mechanisms.
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PMID:Attachment and ingestion of mycoplasmas by mouse macrophages. I. Kinetics of the interaction and effects on phagocyte glucose metabolism. 85 Nov 67

M. gallisepticum membranes were treated with 0.3M lithium diiodosalicylate (LIS) and, on average, 43% of the original membrane proteins were extracted. The extract contained particles with a sedimentation coefficient of 13S and some aggregated proteins. This LIS extract was immunogenic, stimulating the production of haemagglutination-inhibiting, growth-inhibiting and precipitating antibodies in rabbits. It was devoid of haemagglutinating (HA) activity for chicken erythrocytes but did inhibit the HA activity of membranes of M. gallisepticum. This inhibitory activity was destroyed by periodate and trypsin, but not by heat. By sedimentation equilibrium in a caesium chloride gradient, the LIS extract was separated into a lipoprotein-like and a glycoprotein fraction. The lipoprotein-like fraction contained the majority of the proteins present in the original extract, had HA activity and blocked antibody which inhibits haemagglutination. These activities were apparently due to the protein moiety, since they were not removed by extraction with n-butanol. The lipoprotein-like fraction behaved similarly to the unfractionated LIS extract in immunodiffusion tests and polyacrylamide gel electrophoresis, producing one periodic acid-Schiff positive band in the latter. The glycoprotein fraction consisted of about 66% carbohydrate and 33% protein. The sugar components were identified as glucose, galactose, glucosamine, galactosamine, glucuronic acid. The glycorprotein fraction did not possess HA but blocked the HA activity of M. gallisepticum membranes. In immunodiffusion it produced one faint precipitation band. The possible significance of glycoprotein in mycoplasma membranes has been discussed.
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PMID:Dissociation of Mycoplasma gallisepticum membranes with lithium diiodosalicylate and isolation of glycoprotein. 121 14

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.
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PMID:Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures. 125 8

Cell surface hydrophobicity of Mycoplasma hyopneumoniae was evaluated by phase partitioning in a hydrocarbon-aqueous mixture, by hydrophobic interaction chromatography, and by salting out with ammonium sulfate. Results obtained by use of these techniques gave evidence that the cell surface of M hyopneumoniae is weakly hydrophobic, compared with strongly hydrophobic Staphylococcus aureus Cowan I and hydrophilic Klebsiella pneumoniae. After treatment of the organisms with trypsin, M hyopneumoniae became less hydrophobic as measured by hydrophobic interaction chromatography. Significant changes in hydrophobicity were not seen after periodate treatment. Electron microscopy of M hyopneumoniae treated with polycationic ferritin revealed an intermediate, compact, unlabeled layer between the cytoplasmic membrane and an external, heavily labeled layer. Electron microscopy of ferritin-labeled M hyopneumoniae after treatment with trypsin or periodate revealed the intermediate layer to be composed of a trypsin-sensitive protein(s). The outer layer was made of periodate-sensitive carbohydrate(s). Therefore, it appears that proteins in the intermediate layer confer at least part of the total hydrophobicity of the mycoplasmal cell and may contribute to adherence of M hyopneumoniae to target respiratory cells by hydrophobic interactions.
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PMID:Morphologic features and hydrophobicity of the cell surface of Mycoplasma hyopneumoniae. 132 25

Monoclonal antibodies (MAbs) were generated against lysates of clinical Mycoplasma hominis isolates. Three of these, designated BG2, BA10, and FE6, recognized an integral membrane protein of M. hominis with an apparent molecular weight of 50,000 (p50). Electron microscopy studies demonstrated that this protein is distributed evenly over the cell surface. These anti-p50 MAbs were species specific for M. hominis; they reacted with 42% of 126 tested clinical M. hominis isolates and showed no reactivity to heterologous mycoplasma species. Immunoblot analysis after limited proteolysis of purified p50 demonstrated that the three MAbs reacted with different epitopes of the protein. Unlike BA10 and FE6, MAb BG2 induced a decrease in arginine metabolism and a reduction of CFU in metabolic inhibition tests. F(ab)2 fragments of MAb BG2 showed the same inhibitory effect as the intact MAb molecule, while Fab and Fc fragments had no influence on vital functions. Preincubation of the mycoplasmas with MAb BG2 followed by trypsin treatment yielded the same amount of CFU as the control without antibodies. In conclusion, the cell aggregates were resolved by the trypsin treatment. These experiments and tests with the antibody fragments led to the conclusion that only the intact MAb structure or the F(ab)2 structure had metabolic inhibition potential and that the observed metabolism inhibition as well as the apparent decrease in viability were a result of agglutination by MAb BG2.
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PMID:Decreased metabolism and viability of Mycoplasma hominis induced by monoclonal antibody-mediated agglutination. 137 Feb 72

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gallisepticum protein band of M(r) approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 micrograms of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. gallisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.
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PMID:Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum. 137 91


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