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 a nd GTP-binding activities of the proteins. Incubation of eIF-2B with [ C-14]NEM results in strong labeling of the 82- and 55-kDa subunits and with less labeling of the other subunits. Preincubation of eIF-2B wit h eIF-2 interferes with [C-14]NEM labeling of the 82- and 55-kDa subun its. All three (alpha, beta, and gamma) subunits of eIF-2 are labeled strongly by [C-14]NEM. Limited digestion of eIF-2B with trypsin inhibi ts nucleotide exchange activity but does not interfere with GTP bindin g, Under these conditions, the 65-kDa subunit is degraded completely w hile the other subunits remain intact. Treatment of eIF-2 with trypsin results in the generation of eIF-2 lacking the beta-subunit (eIF-3 al pha gamma). eIF-2(alpha gamma) binds [H-3]GDP equally well as intact e IF-2. In the presence of eIF-2B, the exchange of [H-3]GDP for GTP from eIF-2.[H-3]GDP prepared with eIF-2(alpha gamma) is diminished conside rably. [H-3]GTP binding to eIF-2(alpha gamma) is also four- to five-fo ld less than to intact eIF-2. In addition, the association of eIF-2B w ith intact eIF-2, but not with eIF-2(alpha gamma), reduces by two-fold the rate and extent of removal of P-32 by alkaline phosphatase from C K-2 phosphorylated 82-kDa subunit. (C) 1996 Academic Press, Inc.