Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

Human thymidine kinase. Purification and properties of the cytosolic enzyme of placenta

Cytosolic thymidine kinase (EC 2.7.1.21) has been purified 5200-fold to apparent homogeneity from normal human placenta. The purification includes sequential affinity chromatography on blue-Sepharose and a thymidine column. The molecular weight of the enzyme determined by gel filtration and sucrose density ultracentrifugation is 92,000. The subunit molecular weight is 44,000, suggesting that the enzyme is a dimer in its native state. With isoelectric focusing, placental thymidine kinase demonstrated a single form with an isoelectric point of 9.1. The final purified enzyme preparation exhibits no immunological cross-reactivity with human mitochondrial thymidine kinase.     

Mapping and identification of the vaccinia virus thymidine kinase gene

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.     

Differences in the induction of thymidine kinase isozymes in estrogen-treated immature and adult rats

Thymidine kinase activity in immature and castrated adult rat uterus has been examined in respose to estrogen treatment. Following estrogen administration. it was found that immature uterine thymidine kinase activity was increased 30-fold after 24 h, but almost no effect was produced on castrated or non-castrated adult uterus. Uterine thymidine kinase activity was separated into three peaks (peak 1, 2 and 3) by means of DEAE-cellulose column chromatography. In response to estrogen, the thymidine kinase isozymes differed in adult and immature uteri. In immature uteri, marked and selective increase of the activity was found in peak I, whereas in adult only a slight increase in peak 2 activity was observed. The thymidine kinase activity in peak 1 and peak 2 were found to have different enzymatic properties and molecular weight, as determined by gel filtration of 125 000 for peak 1 and 100 000 for peak 2.From these results, it is suggested that estrogen induces specific thymidine kinase isozyme in immature uterus and that the isozyme may be involved in DNA synthesis. Such a induction mechanism seems to be lost during the development.     

Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells.

Noninfected and varicella-zoster virus (VZV)-infected human foreskin fibroblasts were examined for thymidine kinase activity. The specific activity of VZV-infected cell extracts was approximately 7.5-fold greater than that of mock-infected cells and 3-fold greater than that of actively growing cells. The pH optimum of VZV-infected cell thymidine kinase activity was found to be 8.0, whereas thymidine kinase activity in noninfected cells exhibited a sharp pH optimum at 7.4. Electrophoretic analysis of cellular enzymes involved in pyrimidine nucleoside phosphorylation revealed at least three enzymes distinguishable by electrophoretic mobility and substrates used. These enzymes were presumed to be thymidine kinase, deoxycytidine kinase, and uridine kinase. The relative mobilities of these enzymes on 5% polyacrylamide gels were 0.18, 0.91, and 0.54, respectively. In VZV-infected cells, a single band of activity catalyzing the phosphorylation of thymidine, deoxyuridine, deoxycytidine, and cytidine was observed with a relative mobility of 0.48. Cellular pyrimidine-phosphorylating enzymes were not detected in VZV-infected cells. The molecular weight of the VZV-induced enzyme was determined to be 72,000 +/- 7%.     

Stabilization of SV40 transformed human fibroblast cytoplasmic thymidine kinase by ATP

Summary Human fibroblast cytoplasmic thymidine kinase is stabilized by ATP. Sedimentation in sucrose gradients shows that in the presence of ATP, cytoplasmic thymidine kinase has a higher molecular weight (54 000) than in the absence of ATP (28 000). Removal of ATP by dialysis results in the loss of enzyme activity. The subsequent addition of ATP restores activity following a second order time course. These results are interpreted to indicate that in a human fibroblast cell line, transformed by SV₄₀ virus, cytoplasmic thymidine kinase is a dimer in the presence of ATP, but a less active monomer in its absence. Mitochondrial thymidine kinase from the same cell line is not affected by ATP.     

Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.     

EFFECTS OF HYPOTHYROIDISM AND UNDERNUTRITION ON DNA CONTENT AND THYMIDINE KINASE ACTIVITY DURING CEREBELLAR DEVELOPMENT IN THE RAT1,2

—The effects of hypothyroidism and several degrees of undernutrition on the development of cerebellar weight, DNA, and thymidine kinase activity were studied in young rats ranging in age from 2 to 22 days. Early propylthiouracil treatment caused a delayed cerebellar cell multiplication. The activity of cerebellar thymidine kinase was suppressed at ages 2 and 5 days and was in excess of control values on days 15 and 22, thus resulting in a delay in the developmental spectrum for thymidine kinase, and extending the time span of activity beyond that of controls. Undernutrition led to varying degrees of reduced cell proliferation at experimental ages 5, 12, and 19 days. Cerebella from the most undernourished animals showed significant differences from controls in thymidine kinase activity at ages 5 and 12 days. Comparisons between sub-groups from within the oversized litters at 5 and 12 days suggested that changes in thymidine kinase activity relate to the degree of undernutrition to which the sub-group is subjected and that during development there may be a critical degree of undernutrition at which a particular essential enzyme becomes affected. This study emphasizes the biochemical similarities and differences between neonatal hypothyroidism and undernutrition, while pointing out the difficulties which exist in biochemical separation of components of the two conditions. Further evidence is presented that thymidine kinase is responsive to hormonal stimuli during cerebellar development and may play an important role in the regulation of DNA biosynthesis in brain as well as other organs.     

Thymidine kinase activity in cardiac muscle during embryonic and postnatal development

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.     

Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor

The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein. Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.     

DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients.

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.     

Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta

Summary Human thymidine kinase TK1 isoenzyme has been purified 1 800-fold from placenta to a specific activity of 2.9 nmoles/min/mg of protein. The rapid purification procedure includes affinity chromatography on a thymidine-Sepharose column. At all stages of purification, the enzyme showed irreversible lability. The native molecular weight was determined to be 45 000. Human placental TK1 exhibited specificity for ATP and thymidine as substrates, and significant inhibition was found only with thymidine nucleotides. TTP was the most effective inhibitor.     

Purification of thymidine kinase by affinity chromatography with an enzyme inhibitor as the ligand

An affinity column for the purification of thymidine kinase is described. The ligand in this column is a glycoprotein isolated from rat kidney. This glycoprotein inhibits phosphorylation of thymidine in cultured cells and in a cell-free assay system. With an affinity column containing the glycoprotein as a ligand, a 24-fold purification of thymidine kinase from an ammonium sulfate fraction of a crude tissue extract can be obtained. Thymidine kinase eluted from the affinity column migrates as one major band on polyacrylamide and as one diffuse major band on sodium dodecyl sulfate-polyacrylamide. The affinity column, with thymidine kinase bound to the inhibitor, can also be used as an assay system. When the glycoprotein is covalently attached to Sepharose, it retains its binding capacity for thymidine kinase but has apparently lost its ability to inhibit the enzyme. Thymidine kinase eluted from the affinity column is again sensitive to the glycoprotein. It seems to be a carbohydrate moiety of the glycoprotein that is responsible for the inhibition.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Human Thymidine Kinase Can Functionally Replace Herpes Simplex Virus Type 1 Thymidine Kinase for Viral Replication in Mouse Sensory Ganglia and Reactivation from Latency upon Explant

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to ~5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.     

Partial purification and characterization of thymidylate kinase from embryonic chick liver.

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10^?5m, and a broad pH optimum between 6.6 and 8.6. A 2–3 mm requirement of Mg²⁺ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate.Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.     

Deoxynucleoside Kinases of Bacillus megaterium KM

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.