A comparison of purified valyl-transfer ribonucleic acid synthetase from <i>Bacillus stearothermophilus</i> and from <i>Escherichia coli</i>

Wilkinson, Susan C.; Knowles, Jeremy R.

doi:10.1042/bj1390391

Cited by 8 publications

(3 citation statements)

References 25 publications

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“…It has been estimated that a free energy of stabilization of less than 3 kcal (ca. 12.6 kJ) per mol can account for the heat stability of most thermophile proteins (18,27). This relatively small energy demand could be met in manifold ways, justifying the currently held view that the're need not be a single general mechanism of thermostabilization for all thermophile proteins.…”

Section: Discussionmentioning

confidence: 99%

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus

Chell¹,

Sundaram²

1978

J Bacteriol

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Malate synthases from a thermophilic Bacillus and Escherichia coli have been isolated in a high state of purity. Molecular weights of these two proteins determined in the native state and after denaturation in sodium dodecyl sulfatemercaptoethanol show that the enzymes are monomeric. This conclusion is supported, for the thermophile enzyme, by the result ofan electrophoretic analysis of that protein after treatment with dimethylsuberimidate and denaturation. The thermophilic Bacillus malate synthase is considerably more thernostable than its mesophilic counterparts from E. coli, Bacillus licheniformis, and Pseudomonas indigofera. It is, however, markedly labilized by an increase in the ionic strength of the medium brought about by the addition of 0.2 M potassium chloride or in pH above 9. Increased ionic strength has little effect on the thermostability of the mesophilic bacterial malate synthases. These observations provide strong support for the idea that monomeric proteins in thermophiles owe their unusual heat stability to the presence of salt bridges in their tertiary structure.

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Section: Discussionmentioning

confidence: 99%

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus

Chell¹,

Sundaram²

1978

J Bacteriol

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“…B. stearothermophilus aminoacyl-tRNA synthetases were assayed as described by Wilkinson & Knowles (1974) except that triethanolamine/HCl buffer, pH7.2, was used instead of cacodylate at the same pH. One unit of enzyme activity is defined as the amount that will incorporate I pmol of '4Clabelled amino acid into aminoacyl-tRNA after min incubation under standard assay conditions.…”

Section: Enzyme Assaymentioning

confidence: 99%

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands

Clarke¹,

Knowles²

1977

Biochemical Journal

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The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.

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“…B. stearothermophilus aminoacyl-tRNA synthetases were assayed as described by Wilkinson & Knowles (1974) except that triethanolamine/HCI buffer, pH7.2, was used instead of cacodylate at the same pH. A unit of enzyme activity is defined as the amount that will incorporate 1,umol of 14C-labelled amino acid into aminoacyl-tRNA after 1 min incubation under standard assay conditions.…”

Section: Enzyme Assaymentioning

confidence: 99%

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand

Clarke¹,

Knowles²

1977

Biochemical Journal

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The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417].

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A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

Cited by 8 publications

References 25 publications

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands

Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand

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