Abstract
The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.
Highlights
The 24-kilodaltonmessenger RNA cap-binding pro- initiation complex (Jagus et al, 1981)
Affinity Purification of Anti-cap-binding protein (CBP) Antibodies-Polyclonal antibodies directed against the 24-kDa CBP, purified from postribosomal supernatant of rabbit reticulocyte lysate by affinity chromatography, were raised in a goat
The immuneserumreactedadditionally, with a 24-kDa polypeptide which co-migrated with CBP
Summary
Of the heme-controlled repressor from rabbit reticulocytes which contained the 24-kDa CBP to generate antibodies. One of these antibodies was claimed to have anti-CBP activity. The antibody inhibited cross-linking to 24-, 28-, 50-, and 80-kDa polypeptides and inhibited translation of (capped) reovirus but not (uncapped) STNV RNA Another monoclonal antibody was prepared using eIF-3 as the immunogen and 24-kDa CBP for screening clones (Sonenberg et ul., 1981b; Sonenberg and Trachsel, 1982). It is evident that these immunological studies have not simplified the question of the polypeptide nature of CBP but have instead added several new candidates. Unlike the monoclonal antibodies discussed above, these antibodies react with the 24-kDa CBP, and only with this polypeptide, suggesting that there areno structurally related higher molecular weight forms
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