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)

The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+-dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+-dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.
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PMID:Functional correlation between cell adhesive properties and some cell surface proteins. 26 20

The formation of a highly organized vascular and corneal endothelial cell monolayer is associated with the appearance of a 60,000-dalton cell surface protein (CSP-60) (30,000 daltons after reduction with dithiothreitol) which is not detectable in rapidly growing endothelial cells and in subconfluent cultures that do not yet exhibit the strict morphology of a confluent monolayer. It is also absent from vascular smooth muscle cells and from endothelial cultures that are maintained in the absence of fibroblast growth factor and grow on top of each other at confluence. After disorganization of cells in a confluent endothelial monolayer by urea, EDTA, or trypsin, CPS-60 is no longer exposed on the cell surface, but it reappears as soon as the cells readopt their characteristic two-dimensional configuration. This reorganization can be achieved in the presence of cycloheximide and despite removal of fibronectin by urea, EDTA, or trypsin. Maximal amounts of fibronectin and no CSP-60 are detected in subconfluent, but not yet organized, endothelial cultures or in endothelial cells that no longer form a monolayer of nonoverlapping cells at confluence. Likewise, cultures of vascular smooth muscle cells contain fibronectin but no CSP-60. These results suggest that CSP-60, rather than fibronectin, could be involved in the adoption of a monolayer configuration by confluent endothelial cells.
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PMID:Appearance in confluent vascular endothelial cell monolayers of a specific cell surface protein (CSP-60) not detected in actively growing endothelial cells or in cell types growing in multiple layers. 28 73

The partially purified glucosyltransferase (GTF) fraction synthesizing primarily water-insoluble glucans, GTF-A, and the homogeneous fraction synthesizing water-soluble glucans, GTF-B, were utilized to assess the binding of GTF activity to the cell surface of Streptococcus mutans GS-5. Growth of the cells in either Todd-Hewitt broth or a chemically defined medium did not appear to affect the ability of the cells to bind either enzyme fraction. Heat inactivation of the cells did not singificantly reduce the interaction of the enzymes with the cells. Cell surface glucan molecules appear to be involved in GTF binding to the cells because: (i) dextranase or alpha-1,3-glucanase treatment of the cells markedly reduced enzyme binding; (ii) the inclusion of soluble dextrans in the binding assays reduced both GTF-A and GTF-B binding to the cells; and (iii) pretreatment of the cells or the GTF-B fraction with soluble dextrans before binding significantly reduced enzyme binding to the cells. In addition, enzyme binding appears to require a cell surface protein component because Pronase, but not trypsin, treatment of cells reduced enzyme binding. Furthermore, the removal of a portion of the cell surface GTF-glucan complex with 3 N NaCl appears to provide additional binding sites for the enzymes. These results are interpreted in terms of the mechanism of the conversion of extracellular GTF to the cell-associated form.
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PMID:Interaction of glucosyltransferase with the cell surface of Streptococcus mutans. 66 17

Previous studies have demonstrated that protease treatment of zona-free mouse eggs impairs sperm-egg interaction (Boldt et al.: Biol Reprod 39:19-27, 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91-101, 1989). In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona-free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 micrograms/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S-methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona-free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm-egg interaction.
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PMID:Recovery of penetration ability in protease-treated zona-free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein. 151 Aug 43

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.
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PMID:Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen. 162 67

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 binds the cell surface protein CD4 with high affinity. Here we report the use of proteolysis to define regions of gp120 involved in CD4 binding. Cleavage of gp120 with Staphylococcus aureus V8 protease at residue 269 or with trypsin at residue 432 destroys CD4 binding. These same sites are protected from proteolytic cleavage by bound CD4. Cleavages at 64, 144, 166, 172, and 315 do not affect binding and are not protected by bound CD4, indicating that these regions are not critical for binding CD4. All proteolytic fragments found in coprecipitates with CD4 were covalently associated via disulfides and comprised complete gp120 molecules. Previous conclusions by Nygren et al. [Nygren, A., Bergman, T., Matthews, T., Jornvall, H. & Wigzell, H. (1988) Proc. Natl. Acad. Sci. USA 85, 6543-6546] that both large and small (95-kDa and 25-kDa) V8 proteolytic fragments bind CD4, independently, are not distinguished by their experiments from the result found here that the small fragment immunoprecipitates with CD4 while disulfide-linked to the larger fragment.
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PMID:CD4-binding regions of human immunodeficiency virus envelope glycoprotein gp120 defined by proteolytic digestion. 176 44

The glycosphingolipids GD3, GM3, and alpha 2, 3-sialosylparagloboside (SPG) are major gangliosides of lymphoid leukemia cells. The reactivity of two monoclonal anti-ganglioside antibodies, an anti-GD3 (R24) and an antibody cross-reactive to GM3 and SPG (M2590), to blasts of patients with T-cell acute lymphoblastic leukemia (ALL) and B-cell precursor ALL (pre-B-ALL), were compared using indirect immunofluorescence and flow cytometry. Results from 23 patients with T-ALL and eight with pre-B-ALL yielded four subclasses of T-ALL and two subclasses of pre-B-ALL. Blasts from most of the patients with T-ALL were R24+M2590- whereas most of the patients with pre-B-ALL were R24-M2590-. Seven of 23 patients with T-ALL had ganglioside immunophenotypes similar to that of pre-B-ALL, i.e. R24-M2590- or R24-M2590+. These subclasses could not be further characterized by additional cell surface immunophenotypic markers or by gene (immunoglobulin and T-cell receptor) rearrangement analysis. The ratio R24/M2590 was less than 1.0 in all patients with pre-B-ALL, and was greater than 1.0 in all patients with T-ALL who were R24 positive, but was not useful in characterizing the double negative T-ALL subclass. To assess whether cryptogenicity of gangliosides due to cell surface protein could account for the low binding of either R24 or M2590, blasts were treated with trypsin before antibody analysis. Whereas the binding of R24 was unchanged after trypsin treatment, binding of M2590 was increased in a number of samples, particularly in those samples which were originally M2590-positive. The results show that comparative staining of T-ALL and pre-B-ALL cells with both anti-GD3 and anti-GM3/SPG antibodies results in a further subclassification of ALL and provides a quantitative assessment of the expression of tumor-associated gangliosides on the blasts of this disease.
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PMID:Immunoreactivity of leukemic lymphoblasts of T-cell and B-cell precursor origin with monoclonal anti-GD3 and anti-GM3 antibodies. 177 57

To examine the mechanism of intercellular adhesion in the establishment of limb skeletal elements we have investigated the process of limb bud cell aggregation in vitro. Limb bud cells are aggregation-competent immediately after their trypsin:collagenase dissociation in the absence of calcium. This aggregation is largely Ca2(+)-independent (CI) and is completely and reversibly inhibited by cycloheximide. In contrast, when limb bud cells are first allowed to recover from Ca2(+)-free trypsin:collagenase dissociation, aggregation of the surviving population is exclusively Ca2(+)-dependent (CD) and completely and reversibly inhibited by cycloheximide. The presence of exogenous calcium during initial cell dissociation retains a functional CD aggregation mechanism. However, incubation of such cells with EGTA releases the CD component and converts the cells to a predominantly CI aggregation. Rabbits were immunized with limb bud cells exhibiting the recovered CD aggregation mechanism and the resulting immune sera were screened for their effect on cell aggregation. Relative to pre-immune sera, intact immune IgG agglutinated dissociated limb bud cells whilst immune Fab fragments inhibited their aggregation. The aggregation-inhibiting antiserum recognizes five major limb bud cell surface components with apparent molecular weights of 72K, 50K, 23K, 14.5K and 8.5K (K = 10(3) Mr), respectively. Limb bud cell surface plasma membranes were isolated by sucrose gradient density centrifugation and detergent-solubilized proteins coupled to Sepharose 4B with cyanogen bromide. Equivalent cell surface plasma membrane proteins were 125I-iodinated and applied to the affinity column. Limb bud cell surface protein affinity chromatography in the presence of exogenous calcium yields a single protein with an apparent molecular weight of approximately 8.5 K. This protein molecule elutes at 0.6 M NaCl, indicating a high affinity, is recognized by the aggregation-inhibiting antiserum, and is itself capable of inhibiting CD limb bud cell aggregation. Fab fragments prepared from rabbit antisera specifically directed against the affinity-purified material also inhibit CD limb bud cell aggregation and this inhibition is neutralized by the 8.5 K protein. Our data thus demonstrate that CD limb bud cell aggregation is not mediated by fibronectin and/or collagen type I and indicate that this process is governed by a novel 8.5 K cell adhesion molecule.
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PMID:An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture. 222 2

Adrenal cortical cells are known to export cAMP and have binding proteins and cAMP-dependent protein kinase activity associated with their plasma membranes. Because these properties suggest a function for extracellular cAMP, we have undertaken a search for specific cell surface receptors for this cyclic nucleotide. Y-1 mouse adrenal tumor cells actively export cAMP by an energy-dependent process. Analysis of Scatchard plots of the equilibrium binding of [3H]cAMP to these cells indicate the existence of two classes of cAMP binders: one with high affinity (ka = 2.9 X 10(9) M-1) and another with low affinity (ka = 7.0 X 10(7) M-1). The cell surface localization of these binders was established by the sensitivity of both the [3H]cAMP-binding proteins and the [32P]8-N3-cAMP photoaffinity labeled proteins of intact cells to mild trypsin digestion and by the surface distribution of a BSA-O2-monosuccinyl cAMP-gold complex revealed by electron microscopy. Analysis of radioautograms of cell surface cAMP-binding proteins from confluent monolayer tumor cells, photoaffinity labeled with [32P]8-N3-cAMP and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major 32P-labeled protein bands which were indistinguishable from the 49,000 and 55,000 mol wt regulatory subunits of the cytosolic protein kinase isoenzymes of this cell. These observations along with the demonstration of cell surface, cAMP-dependent protein kinase activity in the mouse adrenal tumor cell strongly suggest that these cAMP-binding proteins function as regulatory proteins for cell surface protein kinases.
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PMID:Characterization of cell surface adenosine 3',5'-monophosphate-binding proteins in Y-1 mouse adrenal tumor cells. 298 78

Thrombomodulin is an endothelial cell surface protein which complexes with thrombin to accelerate protein C activation. To gain insight into the mechanisms of thrombomodulin-membrane association, limited proteolytic digestions of thrombomodulin with trypsin and elastase were performed. Trypsin digestion resulted in two major fragments (Mr = 54,000 and 27,000), both of which bound to phosphatidylcholine/phosphatidylserine vesicles. Elastase digestion also yielded two major fragments (Mr = 50,000 and 25,000), but only the smaller fragment bound to the phospholipid vesicles. The larger fragment obtained from both enzymatic digestions retained the ability to accelerate protein C activation. The Mr = 54,000 fragment from the trypsin digest retained a high affinity for thrombin (Kd less than or equal to 0.5 nM), a Km for protein C of approximately equal to 8 microM, and a half-maximal Ca2+ dependence of 0.3 mM. The Mr = 50,000 fragment from elastase digestion had a lower affinity for thrombin (Kd approximately equal to 6 nM) than intact thrombomodulin, and the Km for protein C was decreased to 0.3 microM in the presence of 0.3 mM Ca2+. The Ca2+ dependence of protein C activation with the Mr = 50,000 fragment was distinctly different from that of thrombomodulin or the active tryptic fragment. The active elastase fragment exhibited a Ca2+ optimum at 0.3 mM and activity rapidly decreased with further increases in Ca2+. At the Ca2+ optimum, the Km for protein C was similar to that observed on endothelial cell surfaces or with thrombomodulin reconstituted into liposomes. Our data demonstrate that thrombomodulin has one or more membrane-binding domains and that an active soluble form with catalytic activity can be generated by limited proteolytic digestion. Digestion with elastase appears to expose a site on thrombomodulin capable of recognizing the gamma-carboxyglutamic acid domain of protein C (residues 1-44 of the light chain). Whether this is the same site which is exposed on thrombomodulin upon incorporation into phospholipid vesicles (see accompanying manuscript) remains to be determined.
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PMID:Proteolytic formation and properties of functional domains of thrombomodulin. 302 70


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