Effect of temperature on ppp(A2′ p) nA binding protein activities in rabbit reticulocyte lysates and other mammalian extracts

Abstract

Lysates of rabbit reticulocytes and other mammalian cells are known to contain an activity which binds with high specificity ppp(A2′p) 3A,3′-[ 32P]pCp. The binding activity shows a marked dependence on preincubation of lysates at different termperatures (4°C – 45°C). For example, binding was increased 50% by preincubation of rabbit reticulocyte lysates at 37°C for 60 minutes. An identical preincubation of mouse brain extracts results in a greater than 90% loss of binding activity. Fractionation of rabbit reticulocyte lysates into the postribosomal supernatant (PRS) and the ribosomal salt wash (RSW), followed by heparin-agarose column chromatoraphy, showed that with the PRS fraction, most of the binding activity is eluted with 600 mM KCl. With the RSW fraction, more than 50% of the binding activity is eluted with 250 mM KCl. These data suggest that multiple ppp(A2′p) nA binding protein activities exist in mammalian cells.