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.
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.
Ohta
K
K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Toriyama
M
M
Miyazaki
M
M
Murofushi
H
H
Hosoda
S
S
Endo
S
S
Sakai
H
H
D000345:Affinity Labels / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003429:Cross Reactions; D005260:Female; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007106:Immune Sera; D008970:Molecular Weight; D010063:Ovum / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000276:immunology; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012617:Sea Urchins; D008941:Spindle Apparatus / Q000378:metabolism*; D013329:Structure-Activity Relationship
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D007106:Immune Sera; D010445:Peptide Elongation Factors; D012360:RNA, Transfer, Phe; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D019204:GTP-Binding Proteins