The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

J Biol Chem. 1990 Feb 25;265(6):3240-7.

Abstract

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / metabolism
  • Amino Acids / analysis
  • Animals
  • Azides / metabolism
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cross Reactions
  • Female
  • GTP Phosphohydrolase-Linked Elongation Factors / metabolism
  • GTP-Binding Proteins / immunology
  • GTP-Binding Proteins / isolation & purification
  • GTP-Binding Proteins / metabolism*
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Immune Sera
  • Molecular Weight
  • Ovum / metabolism*
  • Peptide Elongation Factors / immunology
  • RNA, Transfer, Phe / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Sea Urchins
  • Spindle Apparatus / metabolism*
  • Structure-Activity Relationship

Substances

  • 8-azidoguanosine triphosphate
  • Affinity Labels
  • Amino Acids
  • Azides
  • Immune Sera
  • Peptide Elongation Factors
  • RNA, Transfer, Phe
  • Guanosine Triphosphate
  • GTP Phosphohydrolase-Linked Elongation Factors
  • GTP-Binding Proteins