Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

Helle Kock Jensen

    Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandling


    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern is a low level of TKl mRNA and TKl enzyme activity in quiescent cells and a high level of TKl mRNA and TKl enzyme in dividing cells. In chronic lymphatic leukemic cells, that are non dividing cells, the results surprisingly showed that the TKl mRNA level was high and at the same level as found in dividing lymphocytes. The high level of TKl mRNA is not translated into active enzyme as the TK enzyme activity was low as in quiescent lymphocytes. These results indicate, that there is a defect in the regulation of TKl in chronic lymphatic leukemic cells. To investigate this phenomenon more closely I decided by recombinant technics to examine the relation between the TKl gene and the TKl protein. In the second part of this investigation a direct expression system for human TKl in E.coli was developed to produce a source of high amounts of TKl, to be able to examine the structure of TKl. The resulting recombinant TKl was similar to native TKl purified from human lymphocytes with regard to sub-unit molecular weight, specific activity, K, for thymidine, K, for ATP, substrate specifity towards AZT and ara-T and native molecular weight as a tetramer. This indicate that the recombinant and native TKl are identical but further investigations showed some interesting differences. Recombinant TKl is about 10 fold more sensitive towards TTP as inhibitor. Furthermore the effect of removal of ATP from the native TKl on the enzyme kinetics and native molecular weight was not found for recombinant TKl. Native TKl shifts reversibly between a low affinity form - a dimer when ATP is absent from the enzyme, to a high affinity form - a tetramer when ATP is present. Recombinant TKl exclusively appear at the high affinity form and only as a tetramer also in the absence of ATP. This indicate that TKl is post-translationally modified in human cells and that this modification can not be performed in E.coli.
    ForlagRoskilde Universitet
    Antal sider118
    StatusUdgivet - 1994

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