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B Munch-Petersen

Publications and source records attributed to B Munch-Petersen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Thymidine in the micromolar range promotes rejoining of UVC-induced DNA strand breaks and prevents azidothymidine from inhibiting the rejoining in quiescent human lymphocytes.

The effect and inter-individual variation in the effect of exogenously added deoxynucleosides (2 x 10(-6) M) on rejoining of UVC-induced DNA strand breaks was examined in quiescent human lymphocytes from 25 healthy persons. Thymidine at concentrations below 2 x 10(-6) M, effectively and with statistically extreme significance, increased rejoining of UVC-induced DNA strand breaks in the lymphocytes of every one of the 25 persons tested (p < 0.0001, Wilcoxon's signed ranks test). The mean stimulation after 20 h of postirradiation repair was 48% (range 18-78%) with an inter-individual variation of 30% (coefficient of variation, CV). Deoxyguanosine stimulated rejoining in 16, but inhibited in three of 19 test persons (mean stimulation 28%, range -31 to 71%). The stimulating effect of deoxyguanosine was also extremely significant (p < 0.0004). Deoxycytidine and deoxyadenosine stimulated rejoining in some persons and inhibited it in others, and without statistical significance (p values above 0.5). The stimulating effect of thymidine was significantly inhibited by deoxycytidine (p < 0.05, n = 12) whereas deoxyguanosine neither promoted or inhibited the stimulation by thymidine (p = 1, n = 12). Rejoining of DNA strand breaks induced by methyl methanesulfonate did not appear significantly stimulated or inhibited by any of the four deoxynucleosides. Finally, the inhibiting effect of azidothymidine (AZT) on rejoining of UVC-induced DNA strand breaks was nullified by the addition of thymidine. In three donors examined, 10(-4) M AZT inhibited the rejoining by about 40-50%. The presence of less than 10(-5) M thymidine reduced the level of UVC-induced DNA strand breaks to below the level in control lymphocytes allowed to repair without AZT. These results indicate that among the four deoxynucleoside triphosphates, dTTP has a crucial role on the repair of UVC-induced DNA damage in quiescent lymphocytes. The results also indicate that an expansion of the dTTP pool may counteract the inhibiting effect of AZT on DNA repair in quiescent lymphocytes.

Adult↗
PubMed1996

Ligands for the affinity chromatography of mammalian thymidine kinase. 1: Strategy, synthesis and evaluation.

Selected thymidine derivatives were synthesized with various spacers and fixed as model compounds at position N-3', C-5, C-3' and C-5', respectively, to simulate the preparation of an affinity gel matrix. Compounds 3, 6, 7 and 9 were evaluated for their effect on pure human cytosolic thymidine kinase (TK). All four compounds showed competitive inhibition with respect to thymidine, with Ki-values between 80 and 1000 microM. In the same positions as the model compounds were bound to the spacers thymidine derivatives were coupled with different Sepharose gel matrices. These affinity matrices were tested for isolation of thymidine kinase out of placental enzyme material. Except for one matrix, more than 98% of the applied activity was retained by the affinity matrices tested. The strongest binding to the enzyme resulted from a fixation at C-5' of the thymidine molecule to the gel matrix.

Binding Sites↗
PubMed1995

Nucleoside triphosphate donors for nucleoside kinases: donor properties of UTP with human deoxycytidine kinase.

The reported higher efficiency of UTP, relative to ATP, as phosphate donor for deoxycytidine kinase (dCK), has been extended and found to apply to both dCyd and dAdo as acceptors. UTP as phosphate donor was shown to follow strictly Michaelis kinetics, with Km = 1 microM, in striking contrast to ATP, which exhibits marked negative cooperativity (Hill coef. = 0.7) with a several-fold higher Kmapp = 15 microM. Phosphate transfer was followed directly with use of mixtures of [gamma-32P]ATP and cold UTP as donors, or with 3H-labeled acceptors and cold donors. With equimolar concentrations of ATP and UTP (50 microM or 1 mM each), and dCyd or dAdo as acceptor, only minimal phosphate transfer occurred from ATP (3-10%). With a 6:1 ratio of ATP:UTP, hence exceeding the intracellular ratio, phosphate transfer from ATP increased, but still did not exceed 25-40% with either dCyd or dAdo as acceptor. Moreover, relative ATP transfer is dependent on the dCyd concentration. We conclude that the major intracellular phosphate donor for dCK is not ATP, but UTP. Preliminary data for human thymidine kinases (TK1 and TK2) exhibit quite different behaviour. The foregoing, together with literature data, are highly relevant to in vitro studies on the properties of the nucleoside kinases, and to the design of chemotherapeutically active nucleoside analogues.

Adenosine Triphosphate↗
PubMed1995

Different affinity of the two forms of human cytosolic thymidine kinase towards pyrimidine analogs.

Recent results showed that ATP enables a kinetically slow shift from a low affinity form to a high affinity form of human cytosolic thymidine kinase (TK1), as reflected by the respective apparent Km values for thymidine of 15 microM and 0.7 microM. The shift is dependent on the concentration of enzyme protein, and calculations indicate that the low affinity form is predominant in G1 cells, and the high affinity form is predominant in S-phase cells. Here, we report that the two forms of TK1 differ manyfold in affinity to the substrate ATP, to the inhibitor dTTP and to various analogs of thymidine substituted in the pyrimidine or sugar. Furthermore, the kinetic reaction mechanisms suggest that the nucleoside analog. 3'-azidothymidine, used for treatment of infections with human immune deficiency virus (HIV), is not a substrate for the low affinity form of TK1.

Adenosine Triphosphate↗
PubMed1995

Human thymidine kinase 1. Regulation in normal and malignant cells.

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.

Adenosine Triphosphate↗
PubMed1995

Increased ratio between deoxycytidine kinase and thymidine kinase 2 in CLL lymphocytes compared to normal lymphocytes.

Deoxycytidine kinase (dCK) is important in the 5'-phosphorylation of deoxynucleoside analogs. Like dCK, thymidine kinase 2 (TK2) catalyzes the initial step of the phosphorylation of dcyd to dCTP. Thymidine is a strong inhibitor of the dCK activity of TK2. We examined the ratio of the dcyd phosphorylation carried out by dCK and by TK2 (dCK/TK2-dcyd) in lymphocytes from CLL patients and from donors. In the CLL lymphocytes we found a 3.5-fold average increase. Therefore, we conclude that addition of thymidine in the treatment of CLL with deoxynucleoside analogs will not be of any advantage. Furthermore, our results can explain earlier findings in CML and AML lymphocytes where the ara-C phosphorylation was twice the dcyd phosphorylation.

Cells, Cultured↗
PubMed1994

Overexpression of human thymidine kinase mRNA without corresponding enzymatic activity in patients with chronic lymphatic leukemia.

The level of cytosolic thymidine kinase (TK1) mRNA in lymphocytes from six healthy people and in lymphocytes from five patients with untreated chronic lymphatic leukemia (CLL) was determined with competitive polymerase chain reaction (competitive PCR). Using this procedure we have shown that in patients with CLL, there is an overexpression of TK1 mRNA without corresponding enzymatic activity. The TK1 mRNA level is approximately 100-fold higher in lymphocytes from CLL patients than in lymphocytes from healthy persons. A high level of TK1 mRNA without corresponding enzyme activity may indicate a defect in the processing of the enzyme. This may disturb the cells' normal feedback system and thereby influence the development of malignant conditions.

Blotting, Northern↗
PubMed1994

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions.

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.

Journal Article↗
PubMed1993

Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with different enzymatic properties.

Human cytosolic thymidine kinase, subunit molecular mass about 24 kDa, is a tetramer in the presence of ATP but a dimer in the presence of thymidine or without substrates. The pure, substrate-free enzyme showed complex, non-hyperbolic thymidine substrate kinetics with an apparent Km of 15 microM. Incubation with ATP at 4 degrees C induced a time-dependent transition to an enzyme form with hyperbolic kinetics and a 20-fold lower Km value for thymidine (0.7 microM) but the same maximal velocity as for cytosolic thymidine kinase (TK1) without ATP. Removal of the ATP by carboxymethyl chromatography reestablished the non-hyperbolic kinetics with the low affinity for thymidine (Km(app) = 12 microM), and this enzyme form could be reversed once more by ATP incubation to the high affinity enzyme form. Similar shifts could not be induced by thymidine. The activating effect of ATP depended on the concentration of enzyme protein in a linear manner. These results indicate that ATP is a positive effector of cytosolic thymidine kinase, controlling a kinetically slow transition between two molecular forms of the enzyme. A hypothetical reaction mechanism is presented to explain the complex kinetic behavior.

Adenosine Triphosphate↗
PubMed1993

UV-induced alterations in skin and lymphocytes during a one-week holiday in the Canary Islands in May.

The effect of solar exposure during a one-week sunbathing vacation in May at 29 degrees N latitude was investigated in 22 volunteers. The following end-points were measured before and after the holiday: skin reflectance at 507 nm, transmission of radiation from 289 nm to 448 nm through the epidermis, epidermal thickness, minimal erythema dose (MED), total white cell, neutrophil, lymphocyte, and lymphocyte subpopulation counts in blood, spontaneous DNA synthesis, DNA strand breaks and sister-chromatid exchange in lymphocytes, and the UVC tolerance of lymphocytes. There was a statistically significant decrease in skin reflectance (p < 0.001) and epidermal transmission (p < 0.01) after the holiday, reflecting increased pigmentation and epidermal thickening. There was a statistically significant increase in epidermal thickness (p < 0.001), MED (p < 0.05), spontaneous DNA synthesis (p < 0.01) and DNA strand breaks in lymphocytes (p < 0.02) after the holiday. The other end-points were not significantly changed. We report that changes in skin pigmentation and epidermal thickness occur after one week of UV exposure. It was also observed that a one-week UV exposure increased both spontaneous DNA repair and the amount of DNA strand breaks in the lymphocytes of the volunteers, while no changes in T-cell subsets were detected.

Adult↗
PubMed1992

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.

Affinity Labels↗
PubMed1991

Diverging substrate specificity of pure human thymidine kinases 1 and 2 against antiviral dideoxynucleosides.

The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.

Antiviral Agents↗
PubMed1991

Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs.

Deoxynucleoside kinases are required for the 5'-phosphorylation of deoxynucleoside analogs used in chemotherapy. Cytoplasmic thymidine kinase (TK1), deoxycytidine kinase (dCK) and mitochondrial thymidine kinase (TK2) were completely purified from human leukemic spleen and their capacities to phosphorylate 43 nucleoside analogs were compared. TK1 showed the most restricted substrate specificity but tolerated 3'-modifications of the sugar ring and some 5-substitutions of the pyrimidine ring. TK2 showed a much broader specificity and phosphorylated pyrimidine bases with bulky 5-substitutions, including cytosine analogs, while sugar analogs with substituents other than OH in the 2' and 3' positions were very poor substrates. dCK showed a very broad specificity phosphorylating several cytosine analogs with 2' and 3' modifications as well as acyclic sugar analogs. Purine deoxyribonucleosides were also efficiently phosphorylated by dCK but in this case sugar modifications led to drastically decreased activity.

Antiviral Agents↗