Purification and characterization of peptide‐elongation factor 2 (aEF‐2) from an extremely halophilic archaebacterium Halobacterium halobium

Publisher Website
Google Scholar
Abstract
A procedure is described for the purification of the archaebacterial peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium. The enrichment was about 530-fold, the obtained preparation practically homogeneous as judged by SDS-PAGE. The poly(U)-dependent poly(Phe) synthesis was completely dependent on aEF-2 in the presence of partially purified aEF-1, and the activity was equivalent to a poly(Phe)-synthesizing system containing unfractionated S-100 enzymes aEF-2 consists of a single peptide with a relative molecular mass of 125000 .+-. 3000 and 100000 .+-. 3000 as determined by SDS-PAGE and gel filtration on Sephadex G-200 respectively. The isoelectric point was 5.7. The amino acid composition analysis indicated the predominance of acidic amino acids (aspartic acid and glutamic acid) and the low content of hydrophobic amino acid (phenylalanine) as compared with those of eukaryotes and prokaryotes. The factor was stable in a pH range from 6 to 8. 2-Mercaptoethanol and GTP but nt GDP markedly protected aEF-2 from heat denaturation at 52.degree.C, aEF-2 became inactivated and insensitive to ADP-ribosylation by diphtheria toxin at low ionic strength but could be renatured by increasing ionic strength. Obviously higher concentrations of salts contribute to the conformational stability of aEF-2.