title: GTPase activity analysis of eRF3 in Euplotes octocarinatus.
abstract: In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were involved in GTP binding.
authors.affiliation: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China.
authors.forename: Li
authors.initials: L
authors.lastname: Song
authors.forename: Jun-Li
authors.initials: JL
authors.lastname: Dong
authors.forename: Ya-Qin
authors.initials: YQ
authors.lastname: Zhao
authors.forename: Bao-Feng
authors.initials: BF
authors.lastname: Chai
authors.forename: Aihua
authors.initials: A
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016812:Euplotes / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases