JBC, Vol. 250, Issue 10, 3660-3665, May, 1975
Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs
P. Voytek
Isoelectric focusing was used as the final step in the isolation of
thymidine phosphorylase which was found to have an isoelectric point of
4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme
preparation contained one major protein band which stained for thymidine
phosphorylase activity and usually a minor, faster migrating band devoid of
activity. Inactivation of thymidine phosphorylase alone or in the presence
of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first
order inactivation kinetics. The rate of inactivation of the enzyme was the
same at pH 5 and 7.4 and the addition of various pyrimidine bases and
nucleosides enhanced the inactivation rate at both pH values, but to a
greater extent at pH 5. Linear plots of inactivation rates versus
concentrations of thymidine or thymine were the same. At 7.8 mM thymidine
or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine
phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves
were obtained with increasing concentrations of either
5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212-
(pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION
OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only
enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH
7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in
sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or
5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates
than the additive inactivation rates of individual compounds, whereas
combinations of either iodo analog with thymidine resulted in lower
inactivation rates. Increasing concentrations of phosphate or NaCl lessened
the photoinactivation rate of thymidine phosphorylase alone and protected
the enzyme from the sensitization caused by the different bases and
nucleosides. No quantitative changes in the number of primary amino groups
in thymidine phosphorylase was evident as a result of irradiation in the
presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination
of the irradiated enzyme on Sephadex G-150 indicated that a larger protein
species is formed and that 5-iodouracil promotes this process.
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