[Natural substrates of uridylylpolynucleotide-(5'P--->O)-tyrosine phosphodiesterase--an enzyme, hydrolyzing the covalent bond between RNA and picornaviral VPg and a synthetic model of them].
A phosphodiester derivative of 3,5-[125I]diiodotyrosine and 5'-[32P]oligodeoxynucleotide mimicking a natural compound--the picornaviral RNA Pg complex--was obtained by the method of active N-hydroxybenzotriazole phosphodiesters. Using the isotope iodine ion exchange reaction in the tyrosine residue, the specific activity of the model compound could be increased 50 times. It has been found that the unlinking enzyme does not hydrolyze the phosphodiester bond between the tyrosine residue and the oligodeoxynucleotide. Treatment of viral RNA-VPg by nuclease S1 gave a 5'-terminal fragment of RNA linked with the K-peptide. This fragment was shown to be split at a higher rate in comparison with the original compound, RNA-VPg. A two-step procedure for obtaining picornaviral RNA-KpA-Kp and RNA-VPg compounds with higher specific activity selectivity labelled at the protein fragment with 125I was developed. At the first stage, aliphatic amino groups of the polypeptide within the RNA-VPg or RNA-Kp complex were selectively acylated by the activated ester of hydroxyphenylpropionic acid, whose residue was iodinated at the second stage by radioactive NaI in the presence of chloramine T. The specific radioactivity of the thus labelled compounds exceeded 15 to 30 times the specific activity upon selective introduction of the label into the protein with the help of the Bolton-Hunter reagent. The method can be used for highly effective and selective introduction of the label into the protein fragment of other nucleoproteins simultaneously at the amino groups of the protein and at tyrosine residue.
Shabanov
A A
AA
Kaliuzhnyĭ
A A
AA
Gottikh
M B
MB
Drygin
Iu F
IuF
D000595:Amino Acid Sequence; D006868:Hydrolysis; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010849:Picornaviridae / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D012367:RNA, Viral; D014764:Viral Proteins; D010727:Phosphoric Diester Hydrolases; C025672:RNA-tyrosine phosphatase