THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Adenosine triphosphate stimulates thymidine incorporation but does not promote cell growth in primary cultures of renal proximal tubule cells.

Acute addition of adenosine triphosphate (ATP) stimulated thymidine incorporation in confluent, quiescent primary cultures of rabbit renal proximal tubule cells in a dose-responsive manner. Similar increases in thymidine incorporation was observed with adenosine diphosphate and adenosine monophosphate but not with adenosine. The effect of chronic administration of ATP, however, suppressed cell growth. This suppression appears to be due to an effect of ATP to cause detachment of cells from culture plates, resulting in an increase in thymidine incorporation acutely but in suppression of cell growth chronically. ATP is, therefore, not a direct growth promoter of renal proximal tubule cells in primary culture.     

The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase

Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex.     

Polyploidy and aspartate-transcarbamylase activity in Hippocrepis comosa L.

The DNA content of plants which were sampled in natural di-, tetra- and hexaploid populations of Hippocrepis comosa L. was estimated and the aspartate transcarbamylase activities of the corresponding cell-free extracts were compared. The amount of DNA is not exactly proportional to the number of genomes. The three kinds of populations do not differ in their aspartate transcarbamylase specific activity. While the enzyme properties are identical in the extracts derived from the diploid and hexaploid plants, the aspartate transcarbamylase present in the tetraploid cytotype shows a slightly lower affinity for one of its substrates and a significantly lower sensitivity to the feedback inhibitor UTP which is still observed after partial purification. These properties might be related to the previously reported greater ability of the tetraploid cytotype to adapt to a variety of biotopes.Abbreviations ATCase aspartate transcarbamylase - CAP carbamylphosphate - EDTA ethylenediaminetetracetic acid - Tris trihydroxymethylaminomethane - AMP adenosine monophosphate - ATP adenosine triphosphate - CMP cytidine monophosphate - CTP cytidine triphosphate - UMP uridine monophosphate - UTP uridine triphosphate     

Phosphorylation of Deoxyribonucleosides and DNA Synthesis during Embryogenesis of the Sea Urchin

The phosphorylation of thymidine, deoxycytidine, deoxyadenosine and deoxyguanosine was studied during the embryogenesis of the sea urchin Hemicentrotus pulcherrimus. [³H]Thymidine was taken up, phosphorylated and accumulated mostly as [³H]thymidine triphosphate in the early cleavage stage embryos. As the embryos developed, the formation of [³H]thymidine triphosphate decreased and most of the [³H]thymidine taken up by the blastulae remained be phosphorylated. When [³H]deoxycytidine was added to the cleaving embryos, the resultant labeled pool consisted of almost equal amounts of [³H]deoxycytidine monophosphate and [³H]deoxycytidine triphosphate. The formation of [³H]deoxycytidine monophosphate increased up to 10 hr following fertilization and then decreased, while the formation of [³H]deoxycytidine triphosphate decreased for 10 hr following fertilization and then gradually increased. [³H]Deoxyadenosine was rapidly phosphorylated to monophosphate derivative in the cleavage stage embryos. The formation of [³H]deoxyadenosine triphosphate increased rapidly after cleavage stage with a concomitant decrease of [³H]deoxyadenosine monophosphate. The activity of phosphorylation in [³H]deoxyguanosine to triphosphate derivative increased rapidly reaching a plateau 10 hr after fertilization. At this point, 80 % of the [³H]deoxyguanosine was recovered as [³H]deoxyguanosine triphosphate. Based on the above results, it was concluded that the profile of production of each deoxyribonucleoside triphosphate changed during the embryogenesis of the sea urchin, and the in vivo rate-limiting step of phosphorylation of the individual deoxyribonucleoside was assumed to be different.     

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.     

Preferential utilization of inorganic polyphosphate over other bioavailable phosphorus sources by the model diatoms Thalassiosira spp.

Polyphosphates and phosphomonoesters are dominant components of marine dissolved organic phosphorus (DOP). Collectively, DOP represents an important nutritional phosphorus (P) source for phytoplankton growth in the ocean, but the contribution of specific DOP sources to microbial community P demand is not fully understood. In a prior study, it was reported that inorganic polyphosphate was not bioavailable to the model diatoms Thalassiosira weissflogii and Thalassiosira pseudonana. However, in this study, we show that the previous finding was a misinterpretation based on a technical artefact of media preparation and that inorganic polyphosphate is actually widely bioavailable to Thalassiosira spp. In fact, orthophosphate, inorganic tripolyphosphate (3polyP), adenosine triphosphate (ATP) and adenosine monophosphate supported equivalent growth rates and final growth yields within each of four strains of Thalassiosira spp. However, enzyme activity assays revealed in all cultures that cell-associated hydrolysis rates of 3polyP were typically more than ~10-fold higher than degradation of ATP and the model phosphomonoester compound 4-methylumbelliferyl phosphate. These results build on prior work, which showed the preferential utilization of polyphosphates in the cell-free exudates of Thalassiosira spp., and suggest that inorganic polyphosphates may be a key bioavailable source of P for marine phytoplankton.     

SYNTHESIS AND PROPERTIES OF NOVEL TRIPHOSPHATE ANALOGUES: RIBONUCLEOSIDE AND DEOXYRIBONUCLEOSIDE (α-P-BORANO, α-P-THIO)TRIPHOSPHATES

The first ribo- and deoxyribo-nucleoside (α-P-borano, α-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (α-P-borano, α-P-thio)triphosphate and thymidine (α-P-borano, α-P-thio) triphosphate have been investigated.     

Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2′-nor-2′-deoxyguanosine

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2′-nor-2′-deoxyguanosine, 2′-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2′-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2′-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2′-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2′-NDG-MP was compared to that of a 2′-NDG-MP preparation obtained by enzymatic phosphorylation of 2′-NDG with HSV-1 thymidine kinase. The results indicated (a) that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and (b) that phosphorylation of 2′-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2′-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.     

Components required for the formation of CH-4 from methylcobalamin by extracts of Methanobacillus omelianskii

Wood, J. M. (University of Illinois, Urbana), and R. S. Wolfe. Components required for the formation of CH(4) from methylcobalamin by extracts of Methanobacillus omelianskii. J. Bacteriol. 92:696-700. 1966.-Optimal conditions for the formation of CH(4) from methylcobalamin (methyl-cobalt-5,6-dimethylbenzimidazolylcobamide) by partially purified extracts of Methanobacillus omelianskii (Methanobacterium omelianskii) were achieved by the addition of two protein fractions, adenosine triphosphate, Mg(++), and a reduced flavin adenine dinucleotide-generating system. Adenosine diphosphate and adenosine monophosphate play an important role in the regulation of C(1) transfer in this reaction. The possible position of adenosine triphosphate in B(12)-dependent methyl transfer is discussed.     

Adenine nucleotides and other factors indicative of glycolytic metabolism in murine spermatozoa

Cyclic adenosine 3':5' monophosphate (cAMP) accumulation during one hour's incubation in 10 mM theophylline and 10 mM pyruvate; initial concentrations of adenosine triphosphate (ATP) and their rate of depletion during one hour's incubation; concentrations of adenosine diphosphate (ADP), adenosine monophosphate (AMP), fructose 2,6 diphosphate (FDP), and glyceraldehyde 3-phosphate (GAP), were assayed in spermatozoa of various genotypes. No effects of transmission ratio distorting t-haplotypes (in heterozygous males) on these variables were found.     

Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Degradation of 5′ adenosine monophosphate in a cell-free system ofEscherichia coli

Sonicated cells ofEscherichia coli contain an enzyme system degrading 5′ adenosine monophosphate (5′ AMP) to hypoxanthine. This enzyme system is located in the fraction sedimenting at 20,000 xg. It has a pH optimum at 8.0. In the fraction sedimenting at 20,000 xg the enzyme activity was inhibited by adenosine triphosphate (ATP). Adenosine and adenine are deaminated by this enzyme preparation to inosine and to hypoxanthine, these activities not being inhibited by ATP.     

Comparative study of metabolism and forms of transport of phosphate between Ascophyllum nodosum and Polysiphonia lanosa

Polysiphonia lanosa (L.) Tandy is a marine red alga that usually grows epiphytically on the fucale Ascophyllum nodosum (L.) Le Jolis. The present work was conducted in order to obtain more information on the relationships between these two algae, especially as regards the metabolism and long-distance transport of phosphorus. Three types of experiments were carried out using labelled phosphorus. (1) Comparative study of the metabolism of ³²P₁ absorbed by the tissues of each species. By means of two-dimensional chromatography and autoradiography, it was shown that ³²P₁ was rapidly incorporated into organic soluble compounds (adenosine triphosphate, hexose monophosphate, uridine diphosphoglucose, phosphoenolpyruvate + phosphoglyceric acid). Although the two algae belong to different phylae the phosphorylated compounds were not very different. The energy charges (0. 72 for both species) were in the usual range for aerobic plant tissues. On the other hand the incorporation of ³²P₁ into the insoluble P₀ fraction was doubled in P. lanosa compared to in A. nodosum (ca 80 and 40%, respectively). At the source level, the air bladder of A. nodosum. the same soluble compounds (inorganic phosphate, P₁ adenosine triphosphate, hexose monophosphate. etc.) represented the likely forms transported. A part of the soluble P₀ fraction may return to the P₁ fraction. (2) In translocation experiments conducted in situ, ³²P₁ locally injected into an air bladder moved over long distances not only through the thallus of A. nodosum but also into P. lanosa. The reciprocal transfer remained unsuccessful. (3) The ³²P₁ represented the predominant compound identified in the two species: this argues in favour of P₁ as the translocated form of phosphorus. Our results support the hypothesis of a parasitic rather than a simple epiphytic relationship between the two algae.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.     

The induction of filopodia in the cellular slime mold Dictyostelium discoideum by cyclic adenosine monophosphate: mechanism of aggregation.

Dictyostelium discoideum vegetative amoebae grown axenically can be induced to extend microprojections, filopodia, in response to cyclic 3′,5′-adenosine monophosphate. Cyclic 3′-5′-guanosine monophosphate, adenosine monophosphate, or adenosine diphosphate at concentrations of 1.0 mm have no effect. After incubation for 15 min, 1.0 mM adenosine triphosphate will also cause filopodial formation. Treatment with 0.1 mM 2–4 dinitrophenol or 1.0 mM sodium azide does not prevent the induction by cyclic adenosine monophosphate. The induced cells can be more extensively agglutinated with Concanavalin A at 0.5 mg/ml than noninduced cells. A model is presented that describes a possible mechanism whereby cells may aggregate via the cyclic adenosine monophosphate induced filopodia.     

Adenosine Triphosphate Content of Selenastrum capricornutum

The adenosine triphosphate content of Selenastrum capricornutum was maintained within 1.4 to 3.4 μg of adenosine triphosphate/mg (dry weight) of living biomass at different growth stages in media of different phosphorus concentrations.     

Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.     

Adenosine Triphosphate and Synchronous Mitosis in Physarum polycephalum

Synchronous mitoses occur in Physarum polycephalum in the absence of cell division. Nucleoside and nucleotide profiles were prepared from synchronously growing P. polycephalum at intervals throughout the growth cycle. Comparison of these profiles demonstrates that the pool of adenosine triphosphate decreases from a high level at prophase to a minimum through mitosis and increases again in the postmitotic period. These events appear to coincide with changes in the pools of adenosine diphosphate and adenosine but not with that of adenosine monophosphate. This observed decrease in the pool of adenosine triphosphate during mitosis was confirmed by direct enzymatic assay. These results presumably reflect the energy demands of the cell during mitosis.