Preparative method of isolating phosphoinositide fractions from brain tissue]

A simple technique for preparative isolation of chromatographically homogenous fractions of mono-, di- and triphosphoinositides from ox brain tissue is described. Podyphosphoinositides fractions were obtained after chromatography of lipid extract on DEAE cellulose, phosphomonoinositides fraction--after chromatography of polyphosphoinositide-free material on aluminium hydroxide column. Bivalent metal ions were eliminated from lipid extract using chromatography on Dowex-50 H+. Ammonium acetate was removed after precipitation of lipids in water: methanol (1:1) in the presence of 4 M this salt. The average yield of mono-, di- and triphosphates was 40, 22 and 58 mg respectively per 1 kg of brain tissue as callulated for lipid phosphorus.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.     

Analysis of nucleotides from Tetrahymena by high-performance liquid chromatography

Procedures for the complete extraction and isocratic high-performance liquid chromatography analysis of cellular nucleoside di- and triphosphate are reported. Maximum nucleotide recovery from cultures of the ciliate Tetrahymena pyriformis is achieved if (a) cells are cooled to 4°C prior to harvest; (b) dicarbonic acid diethyl ester, a protein crosslinking reagent, is added prior to neurtralization of the cellular acid extract; and (c) 350 mm tri-n-octylamine in Freon is used as a neutralizing agent. These procedures also extend the useful lifetime of the Waters μBondapak NH₂ column used for high-performance liquid chromatography analysis. Nucleoside triphosphates are resolved on this column in under 25 min using 125 mm ammonium phosphate, pH 3.15. Nucleoside diphosphates are separated by reducing the buffer concentration to 75 mm.     

Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.     

Possible Interference by an Acid-stable Enzyme during the Extraction of Nucleoside Di- and Triphosphates from Higher Plant Tissues

Acid extracts from tissues of two solanaceous plants were found to contain a heat-labile, nondialyzable factor which hydrolyzes nucleoside di- and triphosphates to nucleoside monophosphates. This acid-resistant factor shows optimal ATP-hydrolyzing activity at pH 5, whereas practically no activity was detected below pH 3 and above pH 9. It does not hydrolyze sugar phosphates, nucleoside monophosphates, uridine diphosphoglucose, and phosphoenolpyruvate. In order to estimate quantitatively the amount of nucleoside di- and triphosphates in a plant extract, care must be taken to circumvent possible interference by this factor. This is achieved by carefully maintaining the extract below pH 3.     

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

Demonstration of separate phosphotyrosyl- and phosphoseryl- histone phosphatase activities in the plasma membranes of a human astrocytoma

A plasma membrane preparation from a human astrocytoma contained p-nitrophenyl phosphate (pNPP), phosphotyrosyl histone, and phosphoseryl histone hydrolysis activities. The pNPPase and phosphotyrosyl histone phosphatase activities were inhibited by vanadate, whereas the phosphoseryl histone phosphatase activity was not; the latter activity was inhibited by pyrophosphate and nucleoside di- and triphosphates. When the membranes were solubilized by Triton X-100 and the solubilized proteins were subjected to column chromatography on DEAE-Sephadex, Sepharose 6B-C1, and wheat germ agglutinin-Sepharose 4B columns, the pNPPase activity from the phosphoseryl histone phosphatase activity. The results from column chromatography also indicated that there may be multiple phosphotyrosyl and phosphoseryl protein phosphatases in the plasma membranes.     

Ribonucleoside uptake and phosphorylation during fertilization and early development of the sea-urchin, Strongylocentrotus purpuratus

Uptake of the four ribonucleosides normally present in RNA increases nearly 50-fold shortly after fertilization in eggs of the sea-urchin, Strongylocentrotus purpuratus. Uridine, adenosine and cytidine are phosphorylated (greater than 95%) to their mono-, di- and triphosphates immediately after transport into the fertilized egg. Although guanosine is transported to an extent equal to the other three ribonucleosides, less than 12% of its phosphorylated after transport. In vitro nucleoside and nucleotide kinase assays of unfertilized egg homogenates indicate that the uridine, adenosine and cytidine kinases as well as the uridylate, adenylate, cytidylate and guanylate kinases are present in the egg prior to fertilization. Substrate competition measurements indicate that adenosine phosphorylation is catalyzed by a monospecific enzyme, while uridine and cytidine phosphorylations are catalyzed by a common kinase. Guanosine kinase activity was not detectable in unfertilized egg homogenates. Between 3 h and 5 h after fertilization the phosphorylation of transported guanosine begins to increase as it enters the embryo. By 7 h after fertilization, more than 95% of the guanosine entering the embryo is phosphorylated to the mono-, di- and triphosphates. More than 80% is phosphorylated to guanosine triphosphate. The timing of increased guanosine phosphorylation correlates with a decrease in the acid-soluble GTP pools in the embryo, suggesting that increased guanosine kinase activity is a response to increased GTP demand. These results, in view of the importance of GTP in many cellular processes, imply a crucial role for guanosine kinase activation in GTP pool maintenance and cellular metabolism during early sea-urchin development.     

A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells

A highly sensitive and rapid assay for the detection of 6-mercaptopurine metabolites in the red blood cells of leukemic patients receiving the drug has been developed. The method employs a batch-chromatographic procedure using a mercurial cellulose resin to selectively absorb thiol compounds combined with separation by high-performance liquid chromatography using a Partisil-SAX column and uv detection. This method permits detection of 6-thioinosine monophosphate, 6-thiouric acid, and 6-thioguanosine mono-, di-, and triphosphates in patient samples with a sensitivity of 5-10 pmol. No 6-thioinosine di- or triphosphates were detected in patient samples. The results of our study indicate that 6-thioguanosine triphosphate is the major metabolite of 6-mercaptopurine retained by red blood cells after oral or iv administration of the drug.     

Facile analysis of ribonucleotides in d,l-α-difluoromethylornithine-treated mouse lymphoid cells by high-performance anion-exchange column chromatography

Using high-performance liquid chromatography with the strong anion-exchange resin column CDR-10 for analysis of ribonucleotides, the effects of d,l-α-difluoromethylornithine (DFMO) on ribonucleotides of mouse leukemic lymphoid cells SC-1 have been studied. More than 16 nucleoside mono-, di and triphosphates, and unknown peaks were clearly separated and measured without increase in baseline rise using the gradient systems of phosphate buffer. The ATP level in DFMO-treated SC-1 cells was reduced, but was reversed by exogenous putrescine. These facts may suggest that polyamine depletion by DFMO during a short time (6 h) caused mitochondrial damage in mammalian cells.     

Facile analysis of ribonucleotides in d,l-α-difluoromethylornithine-treated mouse lymphoid cells by high-performance anion-exchange column chromatography

Using high-performance liquid chromatography with the strong anion-exchange resin column CDR-10 for analysis of ribonucleotides, the effects of d,l-α-difluoromethylornithine (DFMO) on ribonucleotides of mouse leukemic lymphoid cells SC-1 have been studied. More than 16 nucleoside mono-, di and triphosphates, and unknown peaks were clearly separated and measured without increase in baseline rise using the gradient systems of phosphate buffer. The ATP level in DFMO-treated SC-1 cells was reduced, but was reversed by exogenous putrescine. These facts may suggest that polyamine depletion by DFMO during a short time (6 h) caused mitochondrial damage in mammalian cells.     

Separation and some properties of two acid phosphatases from suspension cultures ofIpomoea sp.

Two acid phosphatases isolated from culturedIpomoea (moring glory) cells were separated by column chromatography on DEAE-cellulose. The two acid phosphatases have different pH optima (pH 4.8–5.0 and 6.0) and do not require the presence of divalent ions. The enzymes possess high activity toward pyrophosphate,p-nitrophenylphosphate, nucleoside di- and triphosphates, and much less activity toward nucleoside monophosphates and sugar esters. The two phosphatases differ from each other in Michaelis constants, in the degree of inhibition by arsenate, fluoride and phosphate and have quantitative differences of substrate specificity. In addition, they also differ in their response to various ions. Issued as NRCC No. 20658     

Selective antiviral agents. The metabolism of 5-propyl-2'-deoxyuridine and effects on DNA synthesis in herpes simplex virus type 1 infections

The metabolism and disposition of 5-propyl-2'-deoxyuridine (Pr-dUrd) in herpes simplex virus type 1 infections were investigated in cell culture using [14C]Pr-dUrd, [32P]orthophosphate, and several methods including high pressure liquid chromatography and isopycnic centrifugation. Results in infected cells indicate Pr-dUrd 1) is taken up and phosphorylated to mono-, di-, and triphosphates; 2) is incorporated into DNA; 3) preferentially inhibits synthesis of viral DNA; 4) blocks re-initiation of viral DNA synthesis even after removal of the nucleoside from the culture; and 5) exerts these effects early in the course of infection (before 6 h postinfection). Pr-dUrd was not phosphorylated in uninfected cells, and had little or no effect on apparent cellular DNA synthesis in infected or uninfected cells. Present evidence suggests one possible antiviral event could be the lethal effect of Pr-dUrd after incorporation into viral DNA by alteration of DNA template-directed functions such as replication.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.     

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for half-maximal velocity and the kinetic order of reaction measured with these phosphate donors. On the contrary, nucleoside di- or triphosphate do not modify the kinetic parameters evaluated for nucleoside acceptors. 5. We suggest that the nucleoside phosphotransferase contains both substrate and regulatory sites. It seems that the free apoenzyme is converted, by means of cooperative interactions between regulatory sites, into an enzyme-nucleotide complex, which is particularly stable at 37 degrees C.     

A proposal for a convenient notation for P-chiral nucleotide analogues. Part 3. Compounds with one nucleoside residue and nonnucleosidic derivatives

Recently, we have proposed a new DP/LP stereochemical notation for P-chiral dinucleoside monophosphate analogues that permits simple correlation between spatial arrangement of the substituents and the configuration at the phosphorus center. As an extension of this work, we present here applications of the DP/LP notation to derivatives containing only one nucleoside unit (e.g., alkyl nucleoside phosphodiesters, nucleoside phosphomonoesters, cyclic phosphate derivatives, nucleoside di-, and triphosphates) and to nonnucleosidic phosphorus compounds.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Nucleotide effects on kinetic properties of mitochondrial ATPase

The hydrolytic activity of mitochondrial ATPase, both in its soluble form as F1-ATPase, or as membrane bound in whole mitochondria, was affected by the presence of free nucleoside di- or triphosphates; these effects were largely depending not only on their concentration but also on the substrate concentration. The existence of a regulatory site or sites is proposed; these sites would have a higher affinity for the free nucleoside triphosphates than for the diphosphates, and the interaction of any of these nucleotides with the proposed regulatory site or sites would lead to an activation. The nucleotide regulatory site or sites seem to be different from the anion binding sites since neither free ATP nor free GTP compete with activating or inhibitory anions.     

Different responses to some stimulators of prolyl hydroxylase activities in various rat organs.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) of soluble fraction (105 000 X g supernatant) of rat granulation tissues was markedly enhanced by addition of nucleoside triphosphates to the assay medium. But the stimulatory activities of nucleoside triphosphates were very different in fractions derived from tissues of rat. In skin, lung or whole fetal tissues other than granuloma, GTP enhanced the enzymatic activity by 3-4 fold. On the other hand, in kidney, liver and spleen tissues it brought about no enhancement. The same results were obtained even if ATP regenerating system was added in the assay medium. The stimulatory effect of nucleoside triphosphates was not seen with the soluble fraction of liver, but it appeared with the enzyme fraction purified by affinity column chromatography. The same phenomenon was observed by addition of bovine serum albumin instead of nucleoside triphosphates as stimulator. We discuss the possible reasons as to why the responses of the enzyme to stimulators were quite different among various tissues.     

A procedure for the isolation and determination of deoxyribose derivatives

A method of determining deoxyribose derivatives in biological material is described. It has high sensitivity, and is particularly useful in that it can be applied to a large range of tissues for which the other available assays are unsuitable. This is because the method is applicable to complex mixtures of nucleotides in which such substances as ribonucleotides are present in very large excess over deoxyribonucleotides, and it is not necessary to equilibrate the nucleotide-precursor pool with radioactive phosphate. The method has mainly been developed with the object of determining deoxyribonucleoside triphosphates, but it can be used to assay ribonucleoside triphosphates, as well as the mono- and diphosphates of both types of nucleoside. The procedure used involves three basic techniques: (1) periodate oxidation and methylamine-induced cleavage of the sugar ring to destroy 2'- and 3'-unsubstituted ribonucleosides; (2) column chromatography to separate the deoxyribonucleotides from each other and from other substances, such as the products of the periodate oxidation; (3) fluorimetric determination of deoxyribose after labilization of the pyrimidine-glycosidic bond by bromination of the heterocyclic ring. Each of these three procedures can be used independently, in conjunction with other analytical procedures.