Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties.

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5'-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic and its activity fluctuates during cell cycle coinciding with the DNA synthesis rate and disappears during mitosis. This fluctuation is important for providing a balanced supply of dTTP for DNA replication.The cell cycle specific activity of TK1 is regulated at the transcriptional level, but posttranslational mechanisms seem to play an important role for the level of functional TK1 protein as well. Thus, the C-terminal of TK1 is known to be essential for the specific degradation of the enzyme at the G2/M phase. In this work, we have studied the effect of deletion of the C-terminal 20, 40, and 44 amino acids of TK1 on in vitro stability, oligomerization, and enzyme kinetics. We found that deletion of the C-terminal fold markedly increased the stability as well as the catalytic activity.

Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties.

Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.

Determination of diethylcarbamazine, an antifilarial drug, in human urine by 1H-NMR spectroscopy.

1H-NMR spectroscopy is a convenient method for determination of diethylcarbamazine (DEC) in urine, and can be used to monitor medication with the drug. Urine samples were mixed with 10% of deuterium oxide as a spectrometer field frequency lock, which is the only sample pretreatment required. Tailored excitation with the 1331 pulse was used for water peak suppression. The quantification of DEC was carried out with the triplet of the N-ethyl group, for which the T1 relaxation time was 1 s. In aqueous solutions, amounts below 1 microgram ml-1 of DEC could be easily detected. In urine, the detectability depended on the level of chemical noise but was better than 10 micrograms ml-1. The accuracy and precision of the method were better than 15%. Analysis of urine from volunteers receiving a single therapeutic dose of DEC (6 mg kg-1 body weight orally) showed that the drug was eliminated in unchanged form during 2 days, in agreement with earlier results. The concentration of DEC in urine several hours after the intake exceeded 100 micrograms ml-1 making the 1H-NMR assay rapid and easy. No significant amounts of the N-oxide of DEC could be detected.

UV-inducible DNA repair in Acinetobacter calcoaceticus.

Bacterial mutation frequency after UV irradiation and phage mutation frequency under conditions of W-reactivation were determined in A. calcoaceticus. With the exception of streptomycin resistance, there was no increase in the frequency of the assayed markers above the background level. The increased survival of phage during W-reactivation was not followed by an increase in the frequency of mutation from turbid to clear plaque formers among phage survivors. The findings suggested that the UV-inducible repair pathway in A. calcoaceticus was error free. Post-irradiation incubation of UV-treated culture before phage infection resulted in a further increase of W-reactivation. As chloramphenicol inhibited this response, it was concluded that de novo protein synthesis was involved in the UV-inducible repair pathway in A. calcoaceticus.

Prophage induction by ultraviolet light in Acinetobacter calcoaceticus.

UV-induction of prophage P78 of Acinetobacter calcoaceticus increased with the UV-dose given to the lysogenic strain from the spontaneous induction frequency of about 0.8% to a maximal frequency of 10%. This 10- to 20-fold increase of induction frequency, as measured by the number of infective centres, was accompanied by a 1000-fold increase in the yield of free phage. This effect was probably due to an increase in burst size under the conditions of lysogenic induction. Unusually, the lysogen was more resistant to UV-irradiation than the corresponding non-lysogenic strain.