Purification and characterization of peptide-elongation factor 2 (aEF-2) from an extremely halophilic archaebacterium Halobacterium halobium.
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 125,000 +/- 3000 and 100,000 +/- 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 not GDP markedly protected aEF-2 from heat denaturation at 52 degrees 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.
Saruyama
H
H
Department of Biology, Faculty of Science, Hokkaido University, Sapporo, Japan.
Sasaki
S
S
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D004167:Diphtheria Toxin / Q000378:metabolism; D006217:Halobacterium / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
D000596:Amino Acids; D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose