@phdthesis{oai:nagoya.repo.nii.ac.jp:00010614,
 author = {西尾, 康二 and Nishio, Koji},
 month = {Nov},
 note = {Aminoacy1-tRNA synthetases are specific for amino acids and tRNAs. The synthetases- are strikingly diverse in their subunit structures α1, α2,α2β2 and α4) and in molecular weights which range from 42K to 380K. In spite of the diversity of molecular weights and subunit structures, the mechanism of aminoacylation of tRNA has been thought to be similar to each other. I am interested in what molecular structure the enzyme has and in how tRNA molecule can be discriminated by its cognate enzyme. In view of these questions, alany1-tRNA synthetase from Bombyx mori was investigated, since the posterior silk glands of Bombyx mori is known to contain abundant amounts of the enzymes specific for glycine, alanine and serine [l,2].  Alany1-tRNA synthetase was purified from the posterior silk glands of the fifth instar of Bombyx mori. The purification procedure comprised ammonium sulfate fractionation and two column chromatographies of DEAF-Sephacel and hydroxyapatite, and yielded about 100 mg of the enzyme per 1 kg of the glands. Values of Km were 800μM for L-alanine, 300μM for ATP and l.4μM for tRNAAla. The pyrophosphate formation of the enzyme was dependent on both of L-alanine and alanine tRNA. This suggests that the enzyme does not function in a ping-pong-mechanism for the substrate binding and product release. The enzyme was proved to be a monomer of l15K dalton by SOS-polyacrylamide gel electrophoresis, gel filtration and dimethy1 suberimidate cross-linking experiments. The possibility that the monomer dimerizes under some conditions (e.g. in the presence of tRNA or at high temperature) was examined, but no evidence for dimerization of the enzyme could be found. The monomeric enzyme and the cognate tRNA formed a 1: 1 complex.  It has been supposed that monomeric aminoacyl-tRNA synthetases with larger than 100K dalton are covalently linked dimers because of their sequence repeats [3-6] and two binding sites for each substrate [7,8]. If this is correct, two active fragments could be isolated from the synthetase by limited tryptic digestion. The enzyme was cleaved into two fragments of 47K and 62K dalton by trypsin. The 47K fragment and the 62K fragment were found to be derived from the N- and C-terminal domains, respectively, of the intact enzyme. One mole of the enzyme was digested to give one mole each of them. The 47K fragment was active in aminoacylation of tRNA, whereas the 62K inactive. Together with the results that the synthetase binds one molecule of alanine tRNA, these results show that the synthetase is not a covalently linked dimer and acts as a structural and functional monomer.  The intact enzyme wats protected from trypsin-attack by only the cognate tRNA. This protection is due to the binding of the tRNA with both of the N- and C-terminal domains. The Km value for tRNA of the N-terminal fragment was 22μM, which was 16-fold higher than that (l.4μM) of the intact enzyme. The molecular activities of the fragment and the intact enzyme were 2.2 s-1 and 16.8 s-1, respectively. These results indicate that the 62K domain binds with tRNA and is responsible for full activity of aminoacylation. The N-terminal donmain must bind with at least the C-C-A terminal region of tRNA molecule, because the 47K fragment was active in tRNA aminoacylation. On the other hand, although I cannot demonstrate what part of tRNA structure is bound to the C-terminal domain, it seems reasonable to state that the C-terminal domain binds to some other regions of tRNA molecule., 名古屋大学博士学位論文 学位の種類:理学博士(論文) 学位授与年月日: 昭和59年11月12日},
 school = {名古屋大学, Nagoya University},
 title = {STRUCTURE AND FUNCTION OF SILKWORM ALANYL-tRNA SYNTHETASE : カイコのアラニルtRNA合成酵素の構造と機能},
 year = {1984}
}