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)

Brain microtubules are found to disperse rods of skeletal muscle myosin and become decorated with amorphous aggregates of myosin. Then microtubules are partially depolymerized by myosin. Myosin shows high Mg2+-GTPase activity which is not influenced by microtubules, and induces the partial depolymerization of microtubules by exhaustion of GTP in the solution. H-meromyosin depolymerizes microtubules like myosin does. H-meromyosin is, however, contaminated with a trace amount of trypsin, which irreversibly depolymerizes microtubules.
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PMID:Depolymerization of brain microtubules by skeletal muscle myosin. 15 54

The digestion of EF-Tu-GDP (or EF-Tu-GTP) by trypsin [EC 3.4.21.4] under native conditions has been shown to proceed through two different and characteristic stages. 1. In the first phase, the protein is transformed into a fragment (Fragment A) with a molecular weight of 39,000 by exposure to trypsin for a relatively short period of time. Fragment A is unable to catalyze the binding of aminoacyl-tRNA to ribosomes. The ability to promote two partial steps of the binding reaction, i.e., formation of the aminoacyl-tRNA-EF-Tu-GTP ternary complex as well as the methanol-stimulated, ribosome dependent GTPase reaction, was rapidly destroyed. On the other hand, the ability to interact with guanine nucleotides as well as EF-Ts survived well during prolonged digestion. 2. In the second phase of digestion, a nick is introduced in Fragment A to yield two subfragments (Fragments B and C). These two fragments exist as a hybrid molecule which migrates as a single peak on a Sephadex G-75 column, and which dissociates into Fragments B and C only in the presence of 6 M guanidine hydrochloride or 5% sodium dodecyl sulfate. The molecular weights of Fragments B and C, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, were 22,000 and 12,000 respectively. The hybrid molecule still retained one mole of bound guanine nucleotide and was resistant to further tryptic digestion. 3. Three sulfhydryl groups of EF-Tu were found to be present in Fragment B, both by amino acid analysis of the purified fragments and also by electrophoresis of tryptic digests labeled with N-ethyl[14C]maleimide. 4. The tryptic digestion of EF-Tu-GDP (or EF-Tu-GTP) labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) at SH2 (the second SH), caused a 30% decrease in the fluorescence emission during the first rapid phase of digestion. This indicates that destruction of the hydrophobic environment near SH2 of EF-Tu occurred in the early phase of tryptic digestion. 5. The kinetic studies on the reaction of ANM with EF-Tu before and after tryptic digestion indicated that both Fragment A and the hybrid molecule reacted with ANM in the presence of GTP three to four times more rapidly than in the presence of GDP. Thus, it appears that the ability to induce conformational transition near SH2 by a change of nucleotide ligands is still retained in the hybrid molecule consisting of Fragments B and C.
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PMID:Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments. 93 63

1. GTPase inhibiting activity for a low M(r) GTP-binding protein, ram p25, was detected in the cytosolic fractions of rat spleen and PC12 cells. 2. The inhibitors were heat-labile and trypsin-sensitive, indicating that they were of proteinous nature. 3. The molecular mass of the inhibitor in the spleen appeared to be about 65 kDa on the elution profile from the gel filtration column.
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PMID:Evidence of the presence of GTPase inhibiting proteins for a low M(r) GTP-binding protein, ram p25, in rat spleen and pheochromocytoma PC12 cells. 145 41

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.
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PMID:A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts. 151 94

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 involvement of the first 69 amino acids of eukaryotic elongation factor 1 alpha (EF-1 alpha) from rabbit reticulocyte in GTP and aminoacyl-tRNA binding has been analyzed by a variety of techniques. EF-1 alpha was subjected to limited trypsin digestion, which cleaved predominantly at residues 36 and 69. A digested form of Escherichia coli EF-Tu, similar to the one used for this study, has been characterized by x-ray crystallography and is used as a structural model for EF-1 alpha. This form of EF-1 alpha bound E. coli Phe-tRNAPhe similar to the wild type protein, but lacked activity in phenylalanine polymerization with poly(U)-programmed ribosomes. These results were obtained regardless of whether or not loosely associated N-terminal peptides were removed by gel filtration chromatography. The digested EF-1 alpha also shows reduced GTPase activity, but the activity is stimulated by both ribosomes and aminoacyl-tRNA. Binding of EF-1 alpha to the 80 S ribosome, as determined by association of reductively methylated protein through Sepharose 6B chromatography, is reduced approximately 7-fold for the limited digested form of the protein. Limited digested EF-1 alpha can, however, be photo-cross-linked with GTP and 3'-p-azido-GTP similar to intact EF-1 alpha. Chemical cross-linking with oxidized GTP, fluorosulfonylbenzoyl-GTP, or with trans-diaminedichloroplatinum(II) and GPT, shows a similar modification of both intact and limited digested EF-1 alpha. In order to further localize the modification site with the GTP reagents and assure that modification was not occurring in the first 69 amino acids, intact EF-1 alpha was modified with these same reagents. Limited trypsin digestion of modified protein indicates that none of these reagents cross-links GTP to the first 69 amino acids of EF-1 alpha, which includes the first GTP binding consensus element, GXXXXGK.
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PMID:Characterization of a limited trypsin digestion form of eukaryotic elongation factor 1 alpha. 199 4

Native elongation factor Tu from Thermus thermophilus is initially attacked by various endoproteases in a region spanning amino acid residues 40-70. By comparing the hydrolysis rates of nucleotide-free and GDP-bound EF-Tu, only a small difference was observed for the tryptic cleavage at Arg-59. Protease V-8 attacks Glu-55 only in a GDP/GTP form, whereas this enzyme exclusively hydrolyze Asn-64 in nucleotide-free EF-Tu, even when the protein had been previously cleaved at Arg-59. Binding of GDP leads to a 42-fold decreased rate of hydrolysis by the Lys-C protease at Lys-52. It also reduces the accessibility of Lys-275 to trypsin, reflecting a "long-range" effect from nucleotide binding domain I to domain II. Only slight differences were observed in the rate of hydrolysis at all positions in the GDP- versus the GTP-bound form. The intrinsic GTPase activity was slightly reduced in trypsin-treated EF-Tu, significantly impaired in EF-Tu cleaved at Lys-52, and completely abolished in EF-Tu cleaved at Asn-64. No ribosome-induced GTPase activity was observed for protease-cleaved EF-Tu's. Treatment of these proteins with periodate-oxidized GDP or GTP followed by cyanoborohydride led to covalent modification of the new N-terminus located exclusively within region 52-60. The highest reactivity was shown by the N-terminus of Glu-56. Additionally, lysine residues in the native protein sensitive to affinity labeling [Peter, M.E., Wittmann-Liebold, B., & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] lost their reactivity upon cleavage of EF-Tu in region 52-60, suggesting an altered structure of the cleaved protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the effector region in Thermus thermophilus elongation factor Tu. 218 98

The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand. Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers. In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins. In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA. Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template. High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein. The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein. We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex. When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex. An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D. M., Silver, L. L., and Nossal, N. G. (1985) J. Biol. Chem. 260, 12851-12857) is also described.
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PMID:Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro. 246 40

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.
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PMID:Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. 249 16


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