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 rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.
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PMID:Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A. 131 Oct 81

Extracts of Vero cells infected with dengue virus type 2 were digested by trypsin in the presence and absence of detergents. The experiments were designed to test the models proposed for flavivirus translation in which the glycoproteins prM, E, and NS1 are inserted into the endoplasmic reticulum of the cell, whereas certain other nonstructural proteins are not. Viral polypeptides were detected by the use of radiolabel, by immunoprecipitation, or by immunoblotting. The results obtained for NS3 and NS5 were as predicted by the models, with membranes providing no protection against digestion by trypsin. Similarly, the results obtained for prM and E were consistent with the models, with membranes protecting against proteolysis. Some molecules of NS1 were protected, while others were sensitive to proteolysis; novel trypsin-resistant fragments of 69,000, 60,000, and 50,000 Mr (all heat-labile), and of 37,000 and 24,000 Mr were detected following treatment of cell extracts with various combinations of trypsin, detergent, and reducing agent. Preliminary experiments suggested that these tryptic fragments are potentially useful in mapping the antigenic epitopes of NS1.
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PMID:The sensitivity of cell-associated dengue virus proteins to trypsin and the detection of trypsin-resistant fragments of the nonstructural glycoprotein NS1. 182 4

The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein, expressed at high levels in neural tissue, which has about 30% homology to ras. Recombinant rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A detergent-soluble factor associated with rat brain membranes exists that accelerates the GTPase activity of both mammalian and recombinant p25rab3A. The activity was thermolabile, sensitive to trypsin, and behaved like an integral membrane protein. GTPase-activating protein (GAP) activity toward p25rab3A was also detected in the cytosolic fraction. This activity was observed in all other tissues examined, in addition to brain. Based upon dose-response data, the rab3A-GAP activity from rat brain was approximately equally distributed between cytosolic and membrane fractions; no activity was found in the nuclear fraction. Recombinant ras-specific GAP had no effect upon the GTPase activity of p25rab3A. By gel filtration chromatography, the factor in rat brain cytosol has a molecular size of 400,000 daltons.
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PMID:Regulation of the GTPase activity of the ras-like protein p25rab3A. Evidence for a rab3A-specific GAP. 184 29

Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action.
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PMID:Role of the C-terminal region of smg p21, a ras p21-like small GTP-binding protein, in membrane and smg p21 GDP/GTP exchange protein interactions. 189 65

The direct binding protein(s) of ras p21 was investigated in the inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody. ras p21 was overlaid on the vesicle proteins immobilized on a nitrocellulose sheet transferred from the gel of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ras p21 bound to bands 4.2 and 6. ras p21-binding to bands 4.2 and 6 was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. Furthermore, when ras p21 was mixed with inside-out vesicles and then centrifuged, ras p21 was coprecipitated with the vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as fat as tested in an in vitro cell-free system.
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PMID:Possible binding proteins of ras p21 in human erythrocyte membrane. 209 62

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.
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PMID:Identification of a nucleotide exchange-promoting activity for p21ras. 211 14

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.
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PMID:Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu. 211 11

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.
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PMID:Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes. 216 11

Invasion of normal tissues is a complex process which requires active locomotion of malignant cells. Recent studies have identified a group of proteins which appear to be specific regulators of cell movement. Various strains and lines of fibroblast-like and vascular smooth muscle cells release into culture medium a unique protein activity which causes contiguous sheets of normal epithelial cells (e.g., Madin-Darby canine kidney, MDCK, cells) to spread and separate into individual cells (i.e., to scatter). Crude conditioned medium and partially purified MDCK scattering activity derived from human iliac artery smooth muscle cells (HIAS) scattered several lines of human squamous carcinoma cells (FaDu and A253) and markedly stimulated migration of carcinoma cells out of multicellular spheroids onto plastic culture surfaces. The scattering activities for MDCK and carcinoma cells showed similar sensitivities to temperature, trypsin treatment, and alteration of pH; both activities were blocked in the presence of cycloheximide. Unlike HIAS-derived factor, a similar MDCK scattering factor derived from ras-transformed NIH 3T3 cells did not scatter human carcinoma cells. These findings indicate that specific normal tissue-derived proteins may affect the mobility of tumor cells. Further studies of such proteins may yield insights into the mechanisms of tumor cell locomotion and tumor invasion.
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PMID:Smooth muscle-derived factor stimulates mobility of human tumor cells. 230 25

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein. 255 24


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