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

J Y Chen,G Kirchner,M Aebi,N C Martin

doi:10.1016/s0021-9258(17)46211-9

J Y Chen, G Kirchner + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)46211-9

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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.

Highlights

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

Summary

RESULTS AND DISCUSSION

Purification of tRNA Nucleotidyltransferae-The tRNA nucleotidyltransferase was purified from S. cerevisiue wild type cells to apparent homogeneity (Fig. 6) according to the protocols described under “Experimental Procedures” Further analysis of fractions obtained from tRNA-Sepharose columns showed that tRNA nucleotidyltransferase activity as measured by CMP incorporation co-fractionates with this M, 59,000 protein (Fig. 7). The multiple step purification from 250 g of yeast cells resulted in a 13% yield of activity. Purification of the enzyme from the overproducer yielded the identical protein (Fig. 8) in better yield and in greater amounts (Table II). It is substantially more abundant in the strain containing the yeast CCA gene on a multicopy plasmid and much more obtained. There are no apparent consequences of overproducing this enzyme to the cell.

Yeast tRNA Nucleotidyltransferae

Ammonium sulfate

Units of activitv