Purification and properties of tRNA nucleotidyl transferase from Musca domestica

The enzyme tRNA nucleotidyl transferase (EC 2.7.7.25) has been highly purified from whole adult houseflies. A molecular weight of 30 000 has been determined. The enzyme requires Mg2+ and tRNA deprived of the 3' terminal sequence CCA for activity in the incorporation of AMP and CMP onto the tRNA. UMP can be incorporated instead of CMP but the latter has a higher affinity than UMP as shown by competition experiments. A complex between the enzyme and tRNA has been shown by sucrose gradient centrifugation, nitrocellulose binding and protection by tRNA against enzyme denaturation at 50 degrees C. Comparative studies with tRNA nucleotidyl transferase purified from larvae, pupae and adult insects indicate that tRNA nucleotidyl transferase from these three developmental stages have the same molecular weight, sedimentation coefficient and optimum pH while the larval enzyme differs from the pupae and adult enzymes in the elution behaviour from a DEAE-cellulose column.