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Modulation of the Feedback Regulation of Thymidine Kinase Activity by pH in 647V Cells¹

Department of Human Oncology, University of Wisconsin Clinical Cancer Center, University of Wisconsin School of Medicine, Madison, Wisconsin 53792

We have studied the effect of pH on the interactions between thymidine kinase, thymidine triphosphate, and 5'-amino-2',5'-dideoxythymidine (5'-AdThd) in purified preparations of the enzyme and in intact 647V cells, a human bladder cancer cell line. Thymidine kinase is competitively inhibited by 5'-AdThd. dTTP feedback inhibits in a noncompetitive fashion. However, 5'-AdThd partially reverses the inhibition produced by dTTP resulting in enhanced enzyme activity. We have found that dTTP (pK_a = 7.5) is a much more potenti inhibitor of purified preparations of thymidine kinase activity at low pH conditions. For example, 2.5 µM dTTP inhibited thymidine kinase activity by 50, 85, and 95% at pH values of 8.0, 7.5, and 6.5, respectively. The interaction of 5'-AdThd (pK_a = 8.5) at either the active (competitive) or the regulatory (deinhibition) site is not altered significantly over a pH range of 6.5 to 9.5. To extend these findings to intact cells, we studied the perturbation of the uptake of thymidine by 5'-AdThd in 647V cells incubated in media buffered at various pH values. In cells exposed to media buffered at pH 8.5 or 7.5, 5'-AdThd maximally stimulated thymidine uptake about 250 and 300% at 10 and 30 µM, respectively. However, at pH 6.5, 300 µM 5' AdThd was required to produce maximal stimulation of about 500%. These observations are consistent with the greater sensitivity of thymidine kinase (in situ) to feedback inhibition by dTTP at the lower pH conditions. Intracellular dTTP pool sizes were not affected by variations in pH during the short time course of our experiments. However, after 1 h, the intracellular concentration of 5'-AdThd was twice that of the extracellular medium in conditions at pH 7.5 and 8.5 but was equimolar across the membrane at pH 6.5. This does not account for the differences in the perturbation of thymidine uptake by 5'-AdThd at various pH values. In general, our results indicate that regulation of thymidine kinase by dTTP is pH dependent, while its modulation by 5'-AdThd is not, and that regulation of thymidine kinase in situ is sensitive to alterations in pH.

¹ Portions of this work have been presented at the American Association for Cancer Research meeting in Atlanta, Georgia, May 1987 (1). Supported by Grant CA 36823.

² Present address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

³ To whom requests for reprints should be addressed, at Pfizer Central Research, Eastern Point Road, Groton, CT 06340.

Received 3/20/89. Revised 7/ 5/89. Accepted 7/17/89.

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