Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver.
Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximately 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2.eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.
Kimball
S R
SR
Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, Hershey, 17033.
Karinch
A M
AM
Feldhoff
R C
RC
Mellor
H
H
Jefferson
L S
LS
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification*; D020662:Guanine Nucleotide Exchange Factors; D008099:Liver / Q000737:chemistry*; D008297:Male; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000737:chemistry
D000911:Antibodies, Monoclonal; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins