Affinity purification and characterization of (2'-5')oligo(adenylate)-dependent RNase from mouse spleen

Murine (2'-5')An-dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2'-5')An. Since the ribonuclease has high affinity to (2'-5')An, optimal non-denaturing conditions were obtained to disrupt the (2'-5')An-nuclease complex. Low-pH buffers in the presence of 0.1% Triton X-100 removed almost 80% of the enzyme from the (2'-5')An-agarose, preserving its (2'-5')An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent crosslinking method and denaturing-renaturing affinity blotting assay. The purified enzyme was a 160-kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver-stained SDS gels. Both a 160-kDa dimer and 78-kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2'-5')An to (2'-5')An-dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4 degrees C. 2-Mercaptoethanol was required to obtain (2-'5')An-binding activity but, interestingly, in the absence of this reducing agent, (2'-5')An-binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2'-5')An. Furthermore, when activated by low amounts of (2'-5')An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2'-5')An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2'-5')An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2'-5')An-binding properties to those identified in spleen extracts.