Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates |
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Authors: | Joseph M Wu Stanley J Wertheimer Behruz Eslami Joanne C Figuereido Biswendu B Goswami |
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Institution: | (1) Department of Biochemistry, New York Medical College, 10595 Valhalla, New York, USA;(2) Department of Molecular Biology, AMC Cancer Research Center, 80214 Lakewood, Colorado, USA |
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Abstract: | Rabbit reticulocyte lysates, gel filtered on Sephadex G-25 with or without ATP (or its analogs), were preincubated at 37°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, - -methylene-ATP, or ATP- -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 gelfiltered lysates, preincubated at 0°C or 37°C for 30 min, showed the following results: (1) all lysates gave a major cross-linking of the radioactive ligand to an 80 000 dalton polypeptide, regardless of the temperature of preincubation, (2) Iysates gel-filtered without ATP, with 0.1 mM ITP, or - -methylene-ATP, showed a significant reduction in the cross-linking of the 80 000 dalton protein, after preincubation at 37°C for 30 min. This decrease was accompanied by an increase in the labeling of two smaller polypeptides.Abbreviations used 2 5 -oligoadenylates
oligonucleotides consisting of 5 -adenylic acid residues joined by a 2 5 -phosphodiester linkage |
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