Immunological detection of the messenger RNA cap-binding protein.
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.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005914:Globins / Q000235:genetics; D007163:Immunosorbent Techniques; D010942:Plant Viruses / Q000235:genetics; D014176:Protein Biosynthesis; D039381:RNA Cap-Binding Proteins; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000737:chemistry
D000906:Antibodies; D002352:Carrier Proteins; D039381:RNA Cap-Binding Proteins; D012333:RNA, Messenger; D005914:Globins