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)

It has previously been shown that mammalian RNA-peptidyl complexes are found in close association with tRNA, but can be separated from the bulk of the tRNA by benzoylated diethylaminoethylcellulose chromatography (Kull, F.J., and Soodak, M. (1971), Biochim. Biophys. Acta 246, l; Gadski, R.A., and Kull, F.J. (1973), Biochemistry 12, 1907). These studies also showed that under aminoacylation conditions the complex fractions were able to act as acceptors for certain amino acids and that the formation of porcine thyroid tyrosyl-complex II was particularly high. Because of this high acceptor function, and because of the importance of tyrosine to thyroid metabolism, further studies were conducted comparing some of the properties of porcine thyroid tyrosyl-complex II with those of porcine thyroid tyrosyl-tRNA. Porcine thyroid tyrosyl-tRNA synthetase was purified in excess of 200-fold and characterized. It was found that maximal aminoacylation was achieved at pH 8.1 in the presence of 150 mM KCl. The Km for tyrosine was determined to be 3.0 X 10(-6) M. The purified thyroid tyrosyl-tRNA synthetase was used under aminoacylation conditions to prepare radioactively labeled porcine thyroid tyrosyl-tRNA and tyrosyl-complex II. Comparisons made using reversed-phase column chromatography (RPC-5) showed distinct differences between the two aminoacylated species and revealed, in addition, a number of isoaccepting forms of tyrosine tRNA. Tyrosyl-complex II was also found to differ from tyrosyl-tRNA in that it is more stable to deacylation at pH 7.0 and at pH 4.4 and to degradation by ribonuclease A. In addition, tyrosyl-complex II, unlike tyrosyl-tRNA, is degraded by trypsin. Ribosomal binding studies showed that tyrosyl-complex II did not respond to the codons for tyrosine, UpApU and UpApC, whereas tyrosyl-tRNA responded to both. It is suggested that thyroid tyrosine complex II is representative of a group of related complexes that constitute the complex II fraction and that, although the complexes resemble tRNA in many respects, they have distinctly different characteristics than conventional tRNA.
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PMID:Thyroid Ribonucleic Acid-Iodopeptides. Comparison of Tyrosyl-Complex II and Tyrosyl-tRNA. 0 30

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff's base through the epsilon-amino group of a lysine within the enzymic active center and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. At alkaline pH, the Schiff's base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which on the other hand leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and of reducing agent concentration on the rate and extent of reduction of the Schiff's base are analysed. Conditions are described (37 degrees C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1:1 covalent complex with tRNAfMet.
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PMID:Complete inactivation and labeling of methionyl-tRNA synthetase by periodate-treated initiator tRNA in the presence of sodium cyanohydridoborate. 4 39

Earlier studies have shown that native phenylalanyl-tRNA synthetase from baker's yeast contains two different kinds of subunits, alpha of molecular weight 73000 and beta of molecular weight 63000. The enzyme is an asymmetric tetramer alpha-2beta-2, which binds two moles of each ligand per mole. Incubation of the purified enzyme with trypsin results in an irreversible conversion: the alpha-subunit remains apparently unchanged but beta is rapidly degraded and yields a lighter species beta of molecular weight 41000. The trypsin-modified enzyme is an alpha-2beta-2 molecule which can still activate phenylalanine but cannot transfer it to tRNA-Phe; furthermore it does not bind tRNA-Phe but its kinetic parameters are identical to those of the native enzyme with respect to ATP and phenylalanine. Therefore the two beta subunits play a critical part in tRNA binding. Isolated alpha or beta subunits exhibit no significant activity and both types of subunit seem to be required for phenylalanine activation.
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PMID:Modification of phenylalanyl-tRNA synthetase from baker's yeast by proteolytic cleavage and properties of the trypsin-modified enzyme. 16 41

Both the aminoacylation and isotopic ATP-PPi exchange activities of native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli are specifically inactivated by incubation in the presence of periodate-treated initiator tRNA Met. The inactivation proceeds through the formation of a reversible Schiff's base between the epsilon-amino group of a lysine within the catalytic center of the enzyme and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. The Schiff's base may be stabilized by reduction with sodium borohydride. Intact tRNA Met f competes with the inactivation by its dialdehyde. It has been verified in the case of the modified enzyme that the protection is afforded according to an equilibrium constant identical to that for tRNA Met f binding at the active site of the enzyme. Finally it is shown that the incorporation of one molecule of the dialdehyde of [14C]tRNA completely destroys the activity of the monomeric trypsin-modified methionyl-tRNA synthetase.
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PMID:Methionyl-tRNA synthetase from Escherichia coli. Inactivation and labeling by periodate-treated initiator tRNA. 22 89

The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5. Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38). Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA).
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PMID:Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex. 23 23

The new form of valyl-tRNA synthetase (EC 6.1.1.9) that appears immediately after infection of Escherichia coli with bacteriophage T4 was purified and subjected to mild proteolysis using five different proteases. The inactivation of aminoacylation activity was both more extensive and rapid than that obtained with valyl-tRNA synthetase purified from uninfected E. coli. The addition of bulk tRNA from E. coli B protected the phage-specific form of valyl-tRNA synthetase from proteolysis, but ATP and valine did not exhibit a similar protective effect. The characteristic property of phage-modified valyl-tRNA synthetase, resistance to denaturation by 4 M urea, remained unaffected during treatment with trypsin. This suggested that the phage-specific factor tau, known to be associated with the synthetase in phage-infected cells, was protected from proteolysis in the synthetase-tau complex. Comparison by isoelectric focusing of normal valyl-tRNA synthetase, the phage-specific form of this enzyme, and phage enzyme from which tau had been removed, revealed no differences in the isoelectric points of these three molecules. Based on these results a model was drawn for the structural changes occurring in valyl-tRNA synthetase after association with the phage factor tau.
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PMID:Analysis of the structure of T4 bacteriophage-modified valyl-tRNA synthetase by limited proteolysis and isoelectric focusing. 33 May 35

Binding of tRNA(Met/f) to the monomeric trypsin-modified methionyl-tRNA synthetase turns off the methionine-dependent isotopic ATP--PPi exchange. In the case of the dimeric native methionyltRNA synthetase, one anticooperatively bound tRNA(Met/f) inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3'-terminal adenosine. Esterification by methionine of the 3' end of tRNA reinforces the affinity of tRNA(Met/f)for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl-tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site. Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNA(Met/f).
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PMID:Interrelation between transfer RNA and amino-acid-activating sites of methionyl transfer RNA synthetase from Escherichia coli. 33 59

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.
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PMID:Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli. 37 61

The nucleotide-free elongation factor from Bacillus stearothermophilus provides a means to study the effect of Mg2+ ions on various reactions of the protein. The binding of GDP to the protein is stimulated by Mg2+. From comparative studies with other metal ions, particularly Mn2+, it appears that this stimulation is due to the formation of a metal - GDP complex which is bound to the protein. Protection against proteolysis by trypsin is afforded by both Mg2+ and Mg - GDP, but not by GDP alone. The rate of substitution of the sulphydryl group associated with aminoacyl-tRNA binding, either 5,5'-dithio-bis(2-nitrobenzoic acid) or N-ethylmaleimide is reduced in the presence of Mg2+ - All these observations show that Mg2+ not only is involved in GDP binding but also has a direct effect on the tertiary structure of the protein.
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PMID:The effect of Mg2+ on some properties of nucleotide-free elongation factor Tu from Bacillus stearothermophilus. 43 34

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.
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PMID:Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland. 56 66


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