This chapter describes the procedures for the purification of the enzyme from Escherichia coli, and rabbit liver. This procedure yields a preparation of the enzyme from rabbit liver that is homogeneous and about 30,000-fold purified. The assay for tRNA nucleotidyltransferase measures the incorporation of radioactive, acid-soluble adenosine triphosphate (ATP) or cytosine triphosphate (CTP) into an acid-insoluble product. Purified preparations of rabbit liver tRNA nucleotidyltransferase contain a poly(C) polymerase activity as an integral part of the protein. This activity can attach long sequences of cytosine monophosphate (CMP) residues to any intact or partially degraded tRNA molecule or to rRNA. The rate of this activity under the usual assay conditions is only about 1% of the normal CMP incorporation and is generally not detected.
ASJC Scopus subject areas
- Molecular Biology