Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia.
Giardia lamblia is totally dependent on salvage synthesis for its pyrimidine requirements. The salvage pathway enzyme, uridine phosphorylase (pyrimidine nucleoside phosphorylase) was purified to apparent homogeneity from G. lamblia crude extracts by fast protein liquid chromatography and gel filtration on a Superose 12 column, resulting in an overall 3500 fold purification and a recovery of 7.5%. Mono P chromatofocusing gave rise to a major activity peak eluting from the column at pH 5.9, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight was found to be 43,000 +/- 2000 from the Superose 12 column, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000 +/- 2000, indicating that it is a monomer. The activities of uridine, deoxyuridine and thymidine phosphorylases from G. lamblia remained associated throughout the purification procedure, suggesting that one enzyme is responsible for the three enzyme activities. The ratio of activities was consistent throughout the purification procedure. In the reverse (anabolic) direction, the enzyme could use both uracil and thymine as substrates. The properties of the phosphorylase differ significantly from those of the mammalian host.
Lee
C S
CS
School of Biochemistry, University of New South Wales, Kensington, Australia.
Jiménez
B M
BM
O'Sullivan
W J
WJ
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005872:Giardia / Q000201:enzymology*; D007700:Kinetics; D010430:Pentosyltransferases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013939:Thymidine Phosphorylase / Q000302:isolation & purification* / Q000502:physiology; D014543:Uridine Phosphorylase / Q000302:isolation & purification* / Q000502:physiology
D010430:Pentosyltransferases; D014543:Uridine Phosphorylase; D013939:Thymidine Phosphorylase