The interaction of rabbit reticulocyte guanine nucleotide exchange factor eIF-2B with chain initiation factor 2: studies with N-ethylmaleimide and trypsin.
Treatment of eIF-2B and eIF-2 with NEM abolishes nucleotide exchange and GTP-binding activities of the proteins. Incubation of eIF-2B with [14C]NEM results in strong labeling of the 82- and 55-kDa subunits and with less labeling of the other subunits. Preincubation of eIF-2B with eIF-2 interferes with [14C]NEM labeling of the 82- and 55-kDa subunits. All three (alpha, beta, and gamma) subunits of eIF-2 are labeled strongly by [14C]NEM. Limited digestion of eIF-2B with trypsin inhibits nucleotide exchange activity but does not interfere with GTP binding. Under these conditions, the 65-kDa subunit is degraded completely while the other subunits remain intact. Treatment of eIF-2 with trypsin results in the generation of eIF-2 lacking the beta-subunit (eIF-2 alpha gamma). eIF-2(alpha gamma) binds [3H]GDP equally well as intact elf-2. In the presence of elf-2B, the exchange of [3H]GDP for GTP from elf-2. [3H]GDP prepared with eIF-2(alpha gamma) is diminished considerably. [3H]GTP binding to eIF-2(alpha gamma) is also four- to five-fold less than to intact eIF-2. In addition, the association of eIF-2B with intact eIF-2, but not with eIF-2(alpha gamma), reduces by two-fold the rate and extent of removal of 32P by alkaline phosphatase from CK-2-phosphorylated 82-kDa subunit.
Singh
L P
LP
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216, USA.
Denslow
N D
ND
Wahba
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology*; D015852:Eukaryotic Initiation Factor-2 / Q000097:blood* / Q000187:drug effects / Q000302:isolation & purification; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology*
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide