Thymidine kinase of rat liver. Activation by phospholipase C and subcellular distribution in the liver

1. The thymidine kinase activity of rat liver is greatly enhanced on addition of phospholipase C to the assay mixture. 2. Most of the thymidine kinase activity of the liver is recovered in the mitochondrial and in the impure ;nuclear' fractions. No activity was detected in purified nuclei prepared in high-density sucrose. 3. A substantial thymidine kinase activity could be detected, with the aid of phospholipase C, in all rat tissues examined.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Identification of true thymidine kinase in Tetrahymena pyriformis ST.

Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg²⁺, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Although the mitochondrial fraction also exhibited ATP-dependent phosphorylation, it is not stimulated by Mg²⁺ and not significantly inhibited by dTTP. Nucleoside phosphotransferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphotransferase activities were separated by polyacrylamide gel electrophoresis, establishing the presence of both enzymes in this organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.     

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.     

Thymidine phosphotransferase and nucleotide phosphohydrolase of the fern Asplenium nidus. General properties and inhibition by adenosine 3'':5''-cyclic monophosphate.

1. Extracts of several plant species contained nucleoside-AMP phosphotransferase activity. The ratio of activity with thymidine to that with uridine as nucleoside substrate was essentially constant, both between species and throughout plant development. Evidence is presented that the total thymidine-AMP phosphotransferase activity of the leaves of Asplenium nidus (bird's-nest fern) and of Helianthus tuberosus (Jerusalem artichoke) increases during maturation. 2. Thymidine-AMP phosphotransferase was purified 22-fold from a very rich source of this activity, extracts of A. nidus. 3. A broad specificity towards both nucleoside and nucleoside 5'-monophosphate substrates is displayed by this preparation, and the evidence suggests that all could be due to a single enzyme. 4. Nucleosides that act as substrates will also inhibit phosphotransfer to other nucleosides, with Ki values close to the corresponding Km values found when utilized as substrates. 5. Ca2+-activated ATP phosphohydrolase was separated from the phosphotransferase by differential complexing to Blue Dextran in the presence of urea, whereas an AMP phosphohydrolase activity was closely associated with thymidine-AMP phosphotransferase through all separation techniques used. 6. Metal ions did not activate either of the latter two activities, and 1,10-phenanthroline was found to inhibit the phosphotransferase. 7. Km values for AMP for the respective activities were 0.11 mM (thymidine phosphotransferase) and 0.20 mM (AMP phosphohydrolase) and for thymidine (phosphotransferase only) 0.88 mM. 8. 3':5'-Cyclic AMP was found to inhibit both phosphotransferase and AMP phosphohydrolase activities, with Ki values of 0.056 mM and 0.15 mM respectively. It is suggested that this inhibitor would be of value in revealing the existence of thymidine kinase in plant extracts with high thymidine phosphotransferase activity.     

Thymidine Phosphorylation in Arachis hypogaea by Combined Activities of Adenosine Triphosphate Phosphohydrolase and Nucleoside Phosphotransferase

Extracts from hypocotyls of germinating peanuts (Arachis hypogaea L.) stimulated the formation of thymidine monophosphate from thymidine and adenosine triphosphate in the presence of magnesium ions. Such extracts were incapable of incorporating isotopic phosphorus from gamma-labelled adenosine triphosphate into thymidine during the synthesis of thymidine monophosphate but were competent in transferring phosphorus from alpha-labelled adenosine triphosphate to thymidine. The apparent thymidine kinase activity thus appeared to result from the combined activities of an adenosine triphosphatase (adenosine triphosphate pyrophosphohydrolase, E. C. 3.6.1.3) and a nucleoside phosphotransferase (E. C. 2.7.1.77). The latter two enzymes could be largely separated by using DEAE-Sephadex chromatography.     

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.     

Correlation of mitochondrial ribonucleotide reductase and thymidine kinase activities with the synthesis of mitochondrial DNA in rat liver during regeneration

3H-thymidine incorporation into DNA of heavy mitochondria from regenerating rat liver and the change of mitochondrial thymidine kinase and ribonucleotide reductase activities are studied in vivo in regenerating rat liver within 6--48 hours after hepatectomy. Synthesis of mitochondrial DNA and changes in the activity of the enzymes studied are found to be undulate. Thymidine kinase activity maxima coincide with those of 3H-thymidine incorporation. Maximal activity of ribonucleotide reductase pre-exists maxima of mitochondrial DNA synthesis.     

The effects of glucocorticoids on thymidine kinase and nucleoside phosphotransferase during development of chicken embryo retina

Thymidine kinase in chick embryo retina reaches its highest values on the 8–10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 × 10^?6M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8–18-day-old chick embryo and, except on the 18th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine kinase during development. Brief periods of exposure to steriods increase nucleoside phosphotransferase activity in isolated chick embryo retinas. When the exposure was longer than 3 h this activity was also clearly decreased. We conclude that other factors are responsible for the natural increment which occurs for this activity during development.     

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids.

Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.     

Effects of partial and sham hepatectomy on ornithine decarboxylase and thymidine kinase activities and mRNA contents

Ornithine decarboxylase and thymidine kinase are enzymes that increase in activity in regenerating liver. We found that both activities and mRNA levels for these enzymes increase significantly after 70% partial hepatectomy in the rat. After sham hepatectomy (laparotomy) there were significant decreases in activity; however, mRNA content was unaltered. Similar decreases in enzyme activity, without changes in mRNA content, were found with pair-feeding, and additional decreases in activity after starvation. In contrast to previous reports of no change in ornithine decarboxylase and thymidine kinase after sham hepatectomy, the present results indicate that decreases occur. This may be mediated by the decrease in food intake after surgery. Dietary factors may be important in the physiologic regulation of these enzymes in the liver.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Herpesvirus proteins: induction of nucleoside phosphotransferase activity after herpes simplex virus infection.

After herpes simplex virus infection of hamster kidney cells there is an induction of nucleoside phosphotransferase activity which can utilize AMP as phosphate donor. The activity is immunologically specific for the infected cell and is induced concomitantly with the virus-coded pyrimidine deoxynucleoside kinase activity. Phosphotransferase activity is not induced in cells lacking both thymidine and deoxycytidine kinase activity.     

Two molecular forms of thymidine kinase in the cytosol of regenerating rat liver.

Two forms (Peak A and Peak B) of thymidine kinase [EC 2.7.1.75] from regenerating rat liver cytosol were resolved and partially purified by Deae-cellulose chromatography. Both fractions were identical with respect to their substrate requirement, pH optima, metal requirements, and molecular weight, as judged by their sedimentation in sucrose density gradient centrifugation. Peak B differed from Peak A in heat sensitivity, inhibition by dCTP and Km for thymidine and ATP. Peak B enzyme was the only enzyme found in normal adult liver and Peak A enzyme was the form increasing predominantly in regenerating liver.     

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Thymidine phosphorylation in synchronous cultures of Tetrahymena pyriformis GL

Recently it has been established that thymidine can be phosphorylated in two ways in Tetrahymena pyriformis

1. 1. by action of thymidine kinase

2. 2. by action of nucleoside phosphotransferase.

The present report confirms that thymidine kinase is a peak enzyme during S phase. It is suggested that a different thymidine concentration in the thymidine kinase assay might explain why previous workers have been unable to find thymidine kinase in Tetrahymena.     

Distribution and Properties of NAD Phosphorylating Reaction

Distribution of NAD phosphorylating reactions, phosphorylation through NAD kinase and phosphotransferase, was investigated. NAD kinase activity was distributed rather widely in bacteria, whereas phosphotransferase activity with p-NPP and NAD was limited to a few genera. Proteus mirabilis showed strong activity of phosphotransferase besides NAD kinase activity.

Partial purification of the phosphotransferase was attempted. The enzyme preparation possessed phosphatase activity as well as phosphotransferase activity. Phosphorylation of NAD proceeded maximally under the conditions below pH 4.0. Cu²⁺ showed stimulating effect on the activity. Besides p-NPP and phenylphosphate, various nucleotides, especially 2′ (or 3′) isomers, served as excellent phosphoryl donors, and various kinds of nucleosides and nucleotides were phosphorylated to form nucleoside monophosphates and nucleoside diphosphates.     

Nucleoside phosphotransferase activity through the growth and cell cycle of Tetrahymena pyriformis GL-I

Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.