Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus.
The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)
Masullo
M
M
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli, Italy.
Raimo
G
G
Parente
A
A
Gambacorta
A
A
De Rosa
M
M
Bocchini
V
V
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001105:Archaea / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006153:Guanosine Diphosphate / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D020648:Peptide Elongation Factor 1; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate