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 ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.
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PMID:Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA. 389 46

It was previously shown that when the tryptic fragment of methionyl-tRNA synthetase from Escherichia coli is incubated with periodate-treated initiator tRNA, it is inactivated due to the formation of a covalent 1:1 complex that could be stabilized by reduction with cyanoborohydride [Hountondji, C., Fayat, G., & Blanquet, S. (1979) Eur. J. Biochem. 102, 247-250]. In this work, the residues labeled in the trypsin-modified enzyme have been identified. After chymotryptic digestion of the protein-tRNA complex, two major labeled peptides (A and B) and a minor one (C) were isolated and identified by sequencing. The radioactivity associated with peptides A-C represented 65-75, 20-25, and 2-4%, respectively, of the radioactivity eluted from the peptide maps. Peptides A and B encompassed lysines-335 and -61, respectively. Both these lysines were fully labeled. Peptide C encompassed lysines-142, -147, and -149, each of which was incompletely labeled. The significance of these results is discussed in light of the known crystallographic structure of the enzyme.
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PMID:Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet. 391 64

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.
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PMID:Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein. 407 79

The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg(++) concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80 degrees C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, amino-acyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.
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PMID:Association factor of ribosomal subunits from Bacillus stearothermophilus. 494 86

Escherichia coli ribosomes washed with 1 M NH(4)Cl were found to function as acceptor for leucine and phenylalanine in the reaction catalyzed by leucyl, phenylalanyl-tRNA:protein transferase. When isolated subunits were acylated with [(14)C]phenylalanine and reisolated by gradient centrifugation, the recovered 30S particles had a specific radioactivity nearly 30 times that of similarly treated 50S particles. Autoradiography of gels, which contained protein from acylated 30S particles, that had been subjected to electrophoresis in 8 M urea and in sodium dodecyl sulfate, suggested that acceptor activity was largely due to a single protein with a molecular weight of about 12,000. Leucine and phenylalanine residues that had been transferred to ribosomal protein were reactive with fluorodinitrobenzene and were released as leucyl- or phenylalanylarginine after treatment with trypsin. The results indicate that leucyl, phenylalanyl-tRNA: protein transferase catalyzes the addition of these amino acids to an NH(2)-terminal arginine residue of a specific ribosomal protein on the 30S subunit.
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PMID:Modification of a specific ribosomal protein catalyzed by leucyl, phenylalanyl-tRNA: protein transferase. 494 16

Alanyl-tRNA synthetase of 115K daltons from Bombyx mori was cleaved into two fragments of 62K and 47K daltons by trypsin. The 47K fragment was active in aminoacylation of tRNA, whereas the 62K fragment was inactive. The 47K and 62K fragments were found to be located at the N- and C-terminal ends, respectively, in the intact enzyme. The intact enzyme was protected from trypsin-attack by the cognate tRNA. The Km value of the 47K fragment for tRNA was 22 microM which is about 16-fold higher than that for the intact enzyme (1.4 microM). The molecular activities of the fragment and the intact enzyme were 2.2 s-1 and 16.8 s-1, respectively. This indicates that the 62K domain enhances affinity for tRNA and it is responsible for the full activity of tRNA aminoacylation. These results do not support the "covalently linked dimer" hypothesis, but indicate that the alanyl-tRNA synthetase is a functional monomer consisting two large domains.
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PMID:Two-domain structure of alanyl-tRNA synthetase from Bombyx mori: isolation of the N-terminal catalytic domain. 609 58

Native elongation factor Tu from Escherichia coli, EF-Tu, is initially attacked by trypsin at three adjacent sites in the primary structure. These are arginine-44, arginine-58, and lysine-56. The rates of hydrolysis at the two arginine residues are about the same but that at the lysine residue is much slower. The products of the tryptic digestion have been analysed by Edman degradation and polyacrylamide gel electrophoresis. The peptide from alanine-45 to arginine-58 is eventually excised and does not complex with the remaining polypeptides (fragments A and D). The loss of this peptide does not lead to a concomitant loss of activity in stimulating polyphenylalanine synthesis. The latter is closely correlated with the further hydrolysis of the remaining fragment (A + D) complex. This complex resembles native EF-Tu in its ability to stimulate both polyphenylalanine synthesis and the binding of aminoacyl-tRNA to 70-S ribosomes, but does not form so stable a ternary complex with aminoacyl-tRNA and GTP as the native protein.
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PMID:Composition and properties of trypsin-cleaved elongation factor Tu. 615 32

Methionyl-tRNA synthetase from Bacillus stearothermophilus, a dimer of molecular weight 2 X 85K, is converted by limited subtilisin digestion into a fully active monomeric fragment of molecular weight 64K. The reversible methionine activation reaction of these enzymes was followed through the variation of the intensity of their trypotophan fluorescence. Equilibrium and stopped-flow experiments show that the rate and mechanism for adenylate formation supported by the monomeric derivative are undistinguishable from those of each adenylating site of the native dimeric enzyme. In contrast, the rate of tRNA aminoacylation is improved upon limited proteolysis of the native enzyme. This behavior can be related to the anticooperativity of the binding of tRNA molecules to native dimeric enzyme. Accordingly, at 25 degrees C, the dimer might behave as a half-of-the-sites enzyme with only one active tRNA site at a time, compared to two after limited proteolysis with consequent irreversible disociation into two 64K fragments. Another modified form of the enzyme is obtained through limited tryptic digestion. This derivative is completely devoid of activity although its molecular weight under nondenaturating conditions remains undistinguishable from that of the 64K fragment generated by subtilisin. Denaturation reveals that this tryptic derivative is composed of two subfragments with molecular weights of 33K and 29K, respectively. The same fragments may also be directly obtained through limited tryptic digestion of the subtilsic fragment. Interestingly, although trypsin treatment has abolished the activity of the enzyme, fluorescence studies demonstrate that the ATP and methionine binding sites have remained intact. It is shown that the effect of the internal cut made by trypsin into the active 64K fragment has been to considerably depress the "coupling" between the methionine and nucleotide binding sites. Finally, the rate of inactivation of the enzyme by trypsin is observed to be substantially decreased by in situ synthetized methionyl adenylate but not by tRNA. These properties and others are discussed in relation to the problem of its significance of repeating sequences and structural "domains" within the class of aminoacyl-tRNA synthetases.
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PMID:Proteolytic cleavage of methionyl transfer ribonucleic acid synthetase from Bacillus stearothermophilus: effects on activity and structure. 625 May 75

A factor which stimulates the aminoacylation of heterologous and homologous tRNAs for lysine and leucine, as well as a mixture of amino acids, has been isolated from cytoplasmic extracts in Drosophila. The stimulatory factor is separated from inorganic pyrophosphatase activity by DEAE-cellulose chromatography and from aminoacyl-tRNA synthetase activity by trichloroacetic acid precipitation. It contains no nucleotidyl transferase activity. It is trypsin-sensitive and heat-stable, indicating that it may be a small protein. Attempts to measure the molecular weight, however, indicate heterogeneity in size, ranging from 20,000 to 65,000. The A53g mutant has four times as much factor Ore-R adults at 0-2 days; by 6-8 days the level has declined to less than one and a half times that of Ore-R. The heightened aminoacylation activity in the mutant extract is accompanied by increased soluble protein levels. It is known that the stimulation of tRNA aminoacylation in A53g is controlled by modifier genes which enhance the expression of the A53g mutant. The possibility that the stimulation factor is a product of the modifier genes is examined.
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PMID:Stimulatory factor for tRNA aminoacylation: possible product of modifier genes in Drosophila melanogaster. 625 67

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.
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PMID:Macromolecular complex of aminoacyl-tRNA synthetases from sheep liver. Identification of the methionyl-tRNA synthetase component by affinity labeling. 628 5


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