Uridine kinase from
Ehrlich ascites tumor cells has been purified about 60,000-fold to apparent homogeneity and with an overall recovery of about 40%. This purification was achieved using
phosphocellulose and
adenosine 5'-triphosphate-agarose affinity chromatography. The subunit molecular mass as judged by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis was 31,000 daltons. With two-dimensional electrophoresis, only one spot was observed, indicating the absence of
isoenzymes. Multiple peaks of activity are routinely observed on ion exchange chromatography or gel filtration, for both crude preparations or homogeneous
uridine kinase, in agreement with our earlier results that this
enzyme exists as multiple interconvertible oligomeric forms (Payne, R. C., and Traut, T. W. (1982) J. Biol. Chem. 257, 12485-12488). The purified
enzyme has a specific activity of 283 mumol/min/mg of
protein at 22 degrees C. Initial velocity studies using
uridine and
ATP are consistent with a sequential mechanism. Km values for
uridine,
cytidine, and
ATP are 40, 57, and 450 microM, respectively.
CTP and
UTP are competitive inhibitors with respect to
ATP, with Ki values for
CTP and
UTP of 10 and 61 microM, respectively. The
enzyme was active with several
nucleoside analogs, the Km values being 69 microM (5-fluorouridine), 200 microM (3-deazauridine), and 340 microM (6-azauridine). The pure
enzyme is very sensitive to freezing, but can be maintained at O degrees C for 8 weeks with only 20% loss of activity. For long-term storage,
enzyme in 50%
glycerol can be maintained at -20 degrees C for many months with no detectable loss of activity.