Purification of Deoxythymidine Kinase by Preparative Disc Gel Electrophoresis and the Effects of Various Halogenated Nucleoside Triphosphates on Its Enzymatic Activity

Abstract

Abstract A 530-fold purification of deoxythymidine kinase from extracts of Escherichia coli B cells has been achieved by the use of preparative polyacrylamide disc gel electrophoresis in the final stages of the purification procedure. Purified preparations of the enzyme contained one major protein band when examined by analytical polyacrylamide disc gel electrophoresis. 5-Iodo-2'-deoxycytidine 5'-triphosphate and 5-bromo-2'-deoxycytidine 5'-triphosphate were more potent allosteric activators of deoxythymidine kinase than the naturally occurring effector 2'-deoxycytidine 5'-triphosphate throughout the pH range of 5.2 to 9.3. Although 5-iodo-2'-deoxyuridine 5'-triphosphate (IdUTP) activated the enzymes at basic pH values, it exerted an inhibitory effect in a manner comparable to 2'-deoxythymidine 5'-triphosphate below pH 6.5. Whereas the inhibitory effect produced by IdUTP at pH 5.4 could not be prevented by even a 12-fold excess of dCTP, the activation caused by IdUTP at pH 7.8 is completely prevented by dTTP. Zone sedimentation studies in sucrose gradients have shown that the sedimentation coefficient of deoxythymidine kinase at pH 7.8 is increased by 5-bromo-2'-deoxycytidine 5'-triphosphate and 5-iodo-2'-deoxycytidine 5'-triphosphate to a greater extent than that produced by dCTP. IdUTP caused an even more marked effect on the sedimentation coefficient observed at pH 5.6 and 7.8 and was similar to that produced by dTTP. Linear reciprocal plots were obtained for varying concentrations of deoxythymidine in the presence of each halogenated effector at pH 5.4 and 7.8. At pH 5.4 IdUTP, like dTTP, was a competitive inhibitor of thymidine phosphorylation. The synthesis of 5-iodo-2'-deoxycytidine 5'-triphosphate from dCTP by direct iodination is described.