Monoclonal antibodies against eIF-2B inhibit translation in rabbit reticulocyte lysate

eIF-2B is a five-subunit 280 kDa protein whose function is to recycle inactive translation factor eIF-2:GDP to its active eIF-2:GTP form through GDP-GTP exchange. To understand the function of individual eIF-2B subunits, they have recently prepared F1, a mouse MAb against highly-purified rabbit eIF-2B. Western analysis shows that F1 recognizes the 82 kDa subunit (epsilon) of eIF-2B either free or in the eIF-2 x 2B complex. F1 inhibits incorporation of /sup 3/H-leucine in hemin-supplemented rabbit reticulocyte lysate with biphasic kinetics with the concomitant loss of polysomes. This inhibition is accompanied by a six to eight-fold decrease in the amount of met-tRNAi in 43S pre-initiation complexes and the appearance of a novel 15S complex. The phosphorylation state of eIF-2 is not altered during F1-dependent translational inhibition. Addition of purified eIF-2B efficiently rescues translation, with reformation of polysomes. eIF-2B catalyzes displacement /sup 3/H-GDP from the /sup 3/H-GDP x eIF-2 binary complex with either GDP or GTP, and this exchange is not inhibited by F1. This suggests an alternative mechanism of inhibition and/or role for eIF-2B in the formation of the eIF-2 x met-tRNAi x GTP ternary complex.