J. Biochem, 1988, Vol. 103, No. 3 508-521
© 1988 Japanese Biochemical Society
research-article |
Peptide Elongation Factor 1 from Yeasts: Purification and Biochemical Characterization of Peptide Elongation Factors l
and 1ß() from Saccharomyces carlsbergensis and Schizosaccharomyces pombe1
* Department of Molecular Biology, School of Science, Nagoya University Chikusa-ku, Nagoya, Aichi 464
** Department of Biochemistry, Primate Research Institute, Kyoto University Inuyama, Aichi 484
2 To whom correspondence should be addressed.
Cytoplasmic elongation factor l
(EF-a) was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 as function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-l was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1ß and EF-1ß
were isolated and highly purified as biologically active entities from the two yeasts. EF-1ßs from the two yeasts have the same molecular weight of 27,000, whereas component of the S. carls. EF-1ß showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-la and EF-1 ß from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-laß) fraction from freshly prepared cell-free extracts of yeast.
1 This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
3 Present address: Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113.