Direct application of radioiodinated aminoacyl tRNA for radiolabeling nascent proteins.
A two-step procedure to incorporate 125I-iodotyrosine into protein synthesized in a reticulocyte lysate is described. In the first step, the iodination of tyrosyl tRNA was catalyzed by a solid-state glycouril compound. More than one-third of 200 microCi of radioiodine became bound to 70 micrograms of aminoacyl tRNA after 15 min at 0 degrees C. The isotope was distributed in a three-to-one ratio of monoiodotyrosine to di-iodotyrosine. In the second step, the soluble product of the radioiodination was transferred directly into a nuclease-treated reticulocyte lysate coded with RNA isolated from the human hepatoma cell line Hep G2. Fractional recovery of radioiodine in nascent protein was maximally 7.6%. Reaction of the product of translation with antibody against alpha-antitrypsin separated an 125I-containing protein having a molecular weight estimated as 47,000. The synthesis of unprocessed alpha-antitrypsin was confirmed by cleavage of the labeled protein with leader peptidase and by its displacement from immunocomplex formation with purified alpha-antitrypsin. The amount of 125I incorporated into alpha-antitrypsin was proportionate to iodinated tRNA additions up to a concentration of 70 micrograms/ml. The synthesis of alpha-antitrypsin as detected in radioautograms after gel electrophoresis was more than twice as sensitive using radioiodinated aminoacyl tRNA as compared with [35S]methionine. Iodine labeling of thyroxine-binding globulin was also demonstrated in the translation product of Hep G2 RNA. Since the specific activity of the radioiodine is high and the means for detection of the isotope efficient, the method described can facilitate the demonstration of quantitatively minor translation products.
D000818:Animals; D007457:Iodine Radioisotopes; D008715:Methionine / Q000378:metabolism; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D051381:Rats; D013462:Sulfur Radioisotopes; D013975:Thyroxine-Binding Proteins / Q000096:biosynthesis; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis
D007457:Iodine Radioisotopes; D012346:RNA, Transfer, Amino Acyl; D013462:Sulfur Radioisotopes; D013975:Thyroxine-Binding Proteins; D000515:alpha 1-Antitrypsin; D008715:Methionine