Direct application of radioiodinated aminoacyl tRNA for radiolabeling nascent proteins
A two-step procedure to incorporate /sup 125/I-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 /sup 125/I-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 /sup 125/I 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 (/sup 35/S)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.
- Research Organization:
- Univ. of Chicago, IL
- OSTI ID:
- 5253788
- Journal Information:
- Anal. Biochem.; (United States), Vol. 2
- Country of Publication:
- United States
- Language:
- English
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PROTEINS
LABELLING
ELECTROPHORESIS
IODINE 125
LABELLED COMPOUNDS
METHIONINE
MOLECULAR WEIGHT
NUCLEASES
RATS
RETICULOCYTES
SULFUR 35
TRACER TECHNIQUES
TRANSFER RNA
TYROSINE
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
ERYTHROCYTES
ESTERASES
EVEN-ODD NUCLEI
HYDROLASES
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MATERIALS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHODIESTERASES
RADIOISOTOPES
RNA
RODENTS
SULFUR ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques