Purification and properties of yeast ATP (CTP):tRNA nucleotidyltransferase from wild type and overproducing cells

Abstract

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) has been purified from wild type cells of the yeast Saccharomyces cerevisiae, as well as from a strain that overproduces the activity. Purification from the wild type strain was accomplished with a multistep protocol including ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and affinity chromatography. The purified enzyme is near homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 59,000 Da is smaller than reported previously. A similar molecular mass is obtained by gel filtration demonstrating that the enzyme is active as a monomer. The pH optimum for the enzyme is around 9.5. The apparent KM values for ATP and CTP were determined to be 5.6 x 10(-4) M and 1.8 x 10(-4) M, respectively. Purification of the enzyme from the overproducing cells was accomplished by a three step protocol with high yield. The nucleotidyltransferase activity from the overproducing cells had a KM for CTP indistinguishable from that of the wild type enzyme, and the mobility of the protein on sodium dodecyl sulfate gels was the same regardless of the source. Thus, the overproducing strain appears to be a good source for large amounts of yeast nucleotidyltransferase for further biochemical and structural studies.