Skip Navigation

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by HARA-YOKOYAMA, M.
Right arrow Articles by MIYAZAWA, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by HARA-YOKOYAMA, M.
Right arrow Articles by MIYAZAWA, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 1984, Vol. 96, No. 5 1599-1607
© 1984 Japanese Biochemical Society

research-article

Purification and Characterization of Glutamyl-tRNA Synthetase from an Extreme Thermophile, Thermus thermophilus HB81

Miki HARA-YOKOYAMA, Shigeyuki YOKOYAMA and Tatsuo MIYAZAWA2

Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113

1 To whom correspondence should be addressed.

Glutamyl-tRNA synthetase has been isolated from an extreme thermophile, Thermus thermophilus HB8. The enzyme has been purified to homogeneity by successive chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite. 11.7 mg of purified enzyme has been obtained from 2 kg of T. thermophilus cells, with a purification factor of 600 with an 11 % yield. From gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme is found to be a monomer protein with a molecular weight of 50,000. The optimum temperature for the aminoacylation of T. thermophilus tRNAGlu is 65°C, and the optimum pH range is 8.0–9.0, in the presence of 5 mM Mg2+. The Km values for ATP, L-glutamate, and T. thermophilus tRNAGlu are 230 µM, 70 µM, and 0.65µM, respectively, in the presence of 50 mM KC1 and 10 mM MgCl2, at pH 8.0 at 65°C. Escherichia coli tRNA2Glu is also a good substrate with a Km value of 0.60 µM at 65°C. The mole fractions of Arg and Leu residues are higher and that of Asx residues is lower than those of E. coli glutamyl-tRNA synthetase. Glutamyl-tRNA synthetase from T. thermophilus is remarkably thermostable; even after incubation for 9 h at 65°C, 70% of the enzyme activity is retained in the absence of any protecting factors. Such an extremely thermostable enzyme with a low molecular weight will be useful for detailed physicochemical analyses on the molecular mechanism of strict recognition by aminoacyl-tRNA synthetases.

1 This work was supported in part by a Grant-in-Aid for Scientific Research (No. 56470126) from the Ministry of Education, Science and Culture of Japan.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 ScienceHome page
O Nureki, D. Vassylyev, K Katayanagi, T Shimizu, S Sekine, T Kigawa, T Miyazawa, S Yokoyama, and K Morikawa
Architectures of class-defining and specific domains of glutamyl-tRNA synthetase
Science, March 31, 1995; 267(5206): 1958 - 1965.
[Abstract] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.