Some properties of 2-5A binding/nucleolytic activities in gel filtered rabbit reticulocyte lysates.
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.
Wu
J M
JM
Wertheimer
S J
SJ
Eslami
B
B
Figuereido
J C
JC
Goswami
B B
BB
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002458:Cell Fractionation; D002850:Chromatography, Gel; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D006358:Hot Temperature; D009843:Oligoribonucleotides / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives
D000227:Adenine Nucleotides; D000345:Affinity Labels; D003911:Dextrans; D009843:Oligoribonucleotides; D012333:RNA, Messenger; C037405:heparin-sepharose; C044356:2',5'-oligoadenyl-5'-diphosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; C025614:sephadex; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease