141c] tRNA … pCpCpA pyrophosphorylase of rat liver

Abstract

This chapter describes the preparation of incubated rat liver transfer RNA (tRNA), in which the pH 5 fraction prepared from the 105,000 g supernatant fraction of rat liver homogenate is incubated for 60 minutes at 37° in the presence of 0.01 M Tris buffer pH 7.4. ethylenediaminetetraacetate (EDTA) 0.1 M pH 8.0 is added to a final concentration of 10 –4 M and 1 volume of water-saturated phenol (freshly redistilled). Whereas in the preparation of pyrophosphorolyzed rat liver tRNA, the pH 5 fraction of rat liver homogenate is incubated for 60 minutes at 37° in the presence of 6 m M inorganic pyrophosphate, 6 m M MgClz, and 40 m M Tris buffer pH 7.6. After the incubation, the solution is adjusted to pH 5 by adding 0.2 N acetic acid; the precipitate, collected by centrifugation, is dissolved in 0.01 M Tris buffer pH 7.6. The incubation in the presence of pyrophosphate and MgCl 2 , as well as the precipitation at pH 5, are repeated, and the precipitate is dissolved in 0.01 M Tris buffer pH 7.6 containing 10 –4 M EDTA. The RNA is then isolated by phenol extraction and alcohol precipitation. Pyrophosphorolyzed tRNA may also be prepared with the aid of tRNA . . . pCpCpA pyrophosphorylase from rat liver. After 2 hours of incubation at 37°, the RNA is extracted with phenol-water, precipitated by ethanol-potassium acetate, and dialyzed against 0.2 M NaC1, followed by dialysis against water. The RNA is then incubated again in the presence of enzyme and pyrophosphate and isolated. The advantage of this method is the absence of nuclease contamination in the purified enzyme and the ability to prepare pyrophosphorolyzed tRNA from different species.