Thymidine kinase (TK) catalyses the ATP-dependent phosphorylation of thymidine to thymidine monophosphate, which is subsequency phosphorylated to thymidine triphosphate and utilized for DNA synthesis. Human cytosolic TK (TKI) is cell cycle regulated, e.g. the TK1 activity increases sharply at the G,-S phase transition and remains elevated throughout S-phase. The regulation of TKI involves transcriptional, post-transcriptional, translational and post-translational regulation mechanisms. In a variety of cancers the regulation mechanisms(s) are changed and TK isoforms with altered biochemical properties have been observed. An investigation of TK1 gene expression will not only provide insight into the regulation mechanism in normal cells but also in cancer cells. Besides, differences in expression, substrate specificity and molecular structure of TKs in healthy and malignant cells can be used for construction of selective nucleoside analogs, only used by cancer TK isoenzymes. In this Ph.D thesis the cell cycle regulated TKI has been subject for two different approaches. 1: Investigation of the relationship between TK1 mRNA level and TK activity in lymphocytes from healthy donors and in lymphocytes from patients with chronic lymphatic leukemia (CLL). 2: Structure-function relationship of recombinant TKI. In the first part a sensitive method (competitive PCR) for quantification of TKI mRNA was established. The TKI mRNA level was quantified in quiescent lymphocytes from control donors (n = 6) and in lymphocytes stimulated to growth by the mitogen phytohemagglutinin. The expression in normal cells was compared with the level of TK1 mRNA level in patients with chronic lymphatic leukemia (n = 5). The results for the six control donors show a very low level of TK1 mRNA (below 0.006~1 O6 copies mg-’ protein) and TK activity (0.009 to 0.01 6 nmol min-’ mg-’ protein) in quiescent lymphocytes. In dividing lymphocytes the TKI mRNA level increases 5 0 t o 5000-fold (3 to 98 x IO6 copies mg-’ protein) with a concomitant increase in TK activity (0.1 2 to 0.76 nmol min-’ mg-’ protein). In CLL cells which are characterized as being quiescent, the TK activity was in the same range as in quiescent lymphocytes from control donors. However, quantification of the TKI mRNA level shows that all five CLL patients had a very high level (6 to 22 x IO6 copies mg-’ protein) of TKI mRNA, corresponding to the level in dividing lymphocytes. As the high TKI mRNA level is not translated into an active enzyme, these results indicate a defect in the regulation of TKI in CLL cells. For the studies of the structure-function relationship of TKI a recombinant TKI protein, which is expressed as a glutathione-S-transferase (GST) fusion protein was used. TKI protein is cleaved from the GST-part with thrombin. Two TKI mutants, TKI-l 93 and TKI-l 76, with deletions from the C-terminal were constructed by the recombinant PCR method. Deletion of 57 amino acids from the C-terminal (TKI- 176) results in an inactive enzyme. Deletion of 40 amino acid from the C-terminal decreases V,,, 2.5-fold (3800 nmol min‘’ mg-’) than the level of the recombinant wildtype (recombinantTK1) which has a V,,, value of 9700 nmol min-’ mg-’. Except for the Vmaxvalue the recombinant TKI and T K I - l 9 3 behave similarly as the native lymphocyte TKI. When ATP is absent from the enzyme, the enzyme appears as a dimer with low affinity for thymidine and when ATP is present the enzyme appears as a tetramer with high affinity for thymidine. K, for thymidine for recombinant TKI and TKI-l93 incubated with ATP is 0.5 *O.l (mean +s.d., n = 3) pM, while enzyme incubated without ATP has a K, of 14.08 20.68 (n = 3) pM for TKI and 12.8 k0.65 (n = 3) ,uM for TKI-193. The Hill coefficient for enzyme incubated with ATP is 1.38 k0.05 (n = 3) for recombinant TKI and 1.25 20.1 5 (n = 3) for TKI-193. For the -ATP forms the Hill coefficient is 0.46 k0.08 (n = 3) for recombinant TKI and 0.5 kO.l (n = 3) for TKI-193. An unchanged affinity for the natural substrate, thymidine, combined with a lower V,,, value suggest that the last 40 amino acids have a structural role or that the ATP binding is disturbed.