Uridine kinase from Ehrlich ascites carcinoma. Purification and properties of homogeneous enzyme.
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.
Payne
R C
RC
Cheng
N
N
Traut
T W
TW
D000818:Animals; D001667:Binding, Competitive; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification*; D012265:Ribonucleotides / Q000494:pharmacology; D013379:Substrate Specificity; D014541:Uridine Kinase / Q000302:isolation & purification* / Q000378:metabolism
D012265:Ribonucleotides; D010770:Phosphotransferases; D014541:Uridine Kinase