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, β-γ-methylene-ATP, or ATP-γ-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 β-γ-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.
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Research Article|
December 01 1985
Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates
Joseph M. Wu;
Joseph M. Wu
1Department of Biochemistry, New York Medical College, Valhalla, New York 10595, USA
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Stanley J. Wertheimer;
Stanley J. Wertheimer
1Department of Biochemistry, New York Medical College, Valhalla, New York 10595, USA
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Behruz Eslami;
Behruz Eslami
1Department of Biochemistry, New York Medical College, Valhalla, New York 10595, USA
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Joanne C. Figuereido;
Joanne C. Figuereido
1Department of Biochemistry, New York Medical College, Valhalla, New York 10595, USA
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Biswendu B. Goswami
Biswendu B. Goswami
2Department of Molecular Biology, AMC Cancer Research Center, Lakewood, Colorado 80214, USA
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Publisher: Portland Press Ltd
Received:
September 23 1985
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 1985 The Biochemical Society
1985
Biosci Rep (1985) 5 (12): 1041–1051.
Article history
Received:
September 23 1985
Citation
Joseph M. Wu, Stanley J. Wertheimer, Behruz Eslami, Joanne C. Figuereido, Biswendu B. Goswami; Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates. Biosci Rep 1 December 1985; 5 (12): 1041–1051. doi: https://doi.org/10.1007/BF01119625
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