Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates

• Published in: Bioscience reports Vol. 5; no. 12; p. 1041
• Main Authors: Wu, J M, Wertheimer, S J, Eslami, B, Figuereido, J C, Goswami, B B
• Format: Journal Article
• Language: English
• Published: England 01-12-1985
• Subjects: Adenine Nucleotides - metabolism; Adenosine Triphosphate - metabolism; Affinity Labels; Animals; Binding Sites; Cell Fractionation; Chromatography, Gel; Dextrans; Electrophoresis, Polyacrylamide Gel; Endoribonucleases - metabolism; Hot Temperature; Oligoribonucleotides - metabolism; Rabbits; Reticulocytes - metabolism; RNA, Messenger - metabolism; Sepharose - analogs & derivatives
• ISSN: 0144-8463
• DOI: 10.1007/BF01119625

Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37 degrees C and their subsequent binding to p3A4,3'-[32P]pCp was studied. Lysates filtered without ATP or in the presence of 0.1 mM 8-bromo-ATP, 1,N6-etheno-ATP, or ITP showed a time-dependent decrease in binding activity. This decrease was completely prevented when lysates were filtered with 0.1 mM ATP, 2'-deoxy-ATP, beta-gamma-methylene-ATP, or ATP-gamma-S. The stability of binding provided by ATP or 2'-deoxy-ATP analogs corresponds to a more active 2-5A dependent endonucleolytic (RNAase L) activity based on studies using [3H] viral mRNA. Chromatography on heparin-agarose showed that ATP-supplemented gel-filtered reticulocyte lysates had a different p3A4,3'-[32P]pCp binding activity elution-profile than lysates gel-filtered in the absence of ATP. Covalent cross-linking of periodate-oxidized p3A4,3'-[32P]pC to gel-filtered lysates, preincubated at 0 degree C or 37 degrees C for 30 min, showed the following results: all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, lysates gel-filtered without ATP, with 0.1 mM ITP, or beta-gamma-methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37 degrees C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.