THE MECHANISM OF FORMATION OF THYMIDINE 5′-TRIPHOSPHATE BY ENZYMES FROM LANDSCHUTZ ASCITES-TUMOUR CELLS

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Abstract
The mechanism of formation of thymidine 5[image]-triphosphate from thymidine by soluble extracts of Landschutz ascites-tumour cells has been examined by an improved method for the complete separation of thymidine nucleotides on columns of ECTEOLA-cellulose. Studies on the time-course of formation of thymidine 5[image]-triphosphate from [3H]thymidine have invariably shown that thymidine 5[image]-monophosphate and thymidine 5[image]-diphosphate may be detected in the reaction mixture very soon after the start of incubation. On no occasion was labelled thymidine 5[image]-triphosphate detected earlier than the diphosphate. In experiments with thymidine 5[image]-[32p] mono phosphate, labelled diphosphate was again detected before labelled triphosphate. When experiments were performed with thymidine 5[image]-diphosphate labelled in either the [alpha]- or [beta]-phosphates with 32p, thymidine 5[image]-triphosphate was formed very rapidly without the intermediate formation of thymidine 5[image]-monophosphate or any higher phosphates of thymidine than the triphosphate. It is concluded that thymidine 5[image]-diphosphate is an intermediate in the formation of thymidine 5[image]-triphosphate from thymidine and thymidine 5[image]-monophosphate.