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.
Stability of the Cross Linking Component of the Biosensor Receptor Layer after Addition of Gold Nanoparticles
