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PubMed1996

Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif.

An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing.

APOBEC-1 Deaminase↗
PubMed1978

Liver growth, biosynthesis of cytidine nucleotides and level of cytochrome P-450 in rat liver after administration of alpha-hexachlorocyclohexane.

The biosynthesis of cytidine nucleotides and the level of microsomal cytochrome P-450 in intact and regenerating rat liver after repeated administration of alpha-hexachlorocyclohexane (alpha-HCH) were compared. In alpha-HCH treated animals the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides is suppressed. In 24-h regenerating liver the incorporation of labelled orotic acid into cytidine nucleotides is markedly activated; the degree of activation is lower in regenerating livers of alpha-HCH treated animals. The changes in the level of cytochrome P-450 vary inversely with the changes in the utilization of [2-14C] orotic acid for the synthesis of cytidine nucleotides. The activity of cytidine triphosphate synthetase of liver cytosol increases shortly after the administration of alpha-HCH; uridine-cytidine kinase is enhanced in the later stages of the drug action. Within 15-45 min after the administration of alpha-HCH the uptake of [U-14 C] cytidine into the liver and its incorporation into RNA cytosine are increased. After the administration of the drug the uptake of [2-14 C] uridine and its incorporation into RNA uracil is also enhanced whereas its utilization for the synthesis of cytidine nucleotides of the acid-soluble extract as well as for the RNA cytosine are suppressed.

Animals↗
PubMed2002

On the formation kinetics of two-dimensional cytidine films.

The kinetics of phase transitions of cytidine adsorbed on mercury are studied by chronoamperometry and capacitance measurements. Cytidine forms highly ordered two-dimensional adlayers in a broad range of pH. In acid solvent, only one kind of condensed layer is formed. In the alkaline solution, cytidine forms two different two-dimensional (2D) adlayers. The minimum capacitance value in adlayer II at pH 5 is 7.0 microF cm(-2) and, at pH 8.3, it is 5.1 microF cm(-2); in adlayer III, the minimum capacitance is 10.6 microF cm(-2). The formation of a physisorbed film of cytidine molecules adsorbed at the mercury surface proceeds by complex mechanisms. From j-t transients, it can be seen that the phase transformations from dilute adlayer Ia to condensed physisorbed film II is accompanied by the reorientation of cytidine molecules at the mercury surface (inverted current transient). The interfacial transformations of the cytidine film yield a sigmoidal C-t transient. This experimentally measured C-t transient were analysed by Avrami theorem. The rate of the transformations from dilute adlayer Ia to condensed film II of cytidine at pH 5 depends strongly on temperature but is only slightly affected by temperature at pH 8.3. The effect of pH and ionic composition of the supporting electrolyte on the rate of transformation of cytidine films was studied as well.

Adsorption↗
PubMedDate not supplied

A quantum chemical study of the catalysis for cytidine deaminase: contribution of the extra water molecule.

Cytidine deaminase is known as an important enzyme responsible for the hydrolytic deamination of cytidine, which is applied as a key step to the conversion of the precursor of the cancer drug to an active form in the living body. Cytidine with water is efficiently converted to uridine with ammonia in the cleft of cytidine deaminase. In this work, the catalysis of cytidine deaminase for the hydrolytic deamination was examined using cytosine as a model of cytidine and the model molecules for the active site of cytidine deaminase by means of the quantum chemical method. We especially investigated the contribution of the water molecule from the solvent to the catalysis, because the X-ray diffraction analysis of a crystal structure has revealed the existence of the water molecule in the vicinity of the substrate bound to the active site inside the cleft. Our computations showed that the extra water molecule from the solvent has a possibility to support the catalysis of cytidine deaminase.

Catalysis↗
PubMed1978

Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite.

The reaction of HeLa-cell 5.8S rRNA with NaHSO3 under conditions in which exposed cytidine residues are deaminated to uridine was studied. It was possible to estimate the reactivities of most of the 46 cytidine residues in the nucleotide sequence by comparing 'fingerprints' of the bisulphite-treated RNA with those of untreated RNA. The findings were consistent with the main features of the secondary-structure model for mammalian 5.85S rRNA proposed by Nazar, Sitz, & Busch [J. Biol. Chem (1975) 250, 8591--8597]. Five out of six regions that are depicted in the model as single-stranded loops contain cytidine residues that are reactive towards bisulphite at 25 degrees C (the other loop contains no cytidine). The cytidine residue nearest to the 3'-terminus is also reactive. Several cytidines residues that are internally located within proposed double-helical regions show little or no reactivity towards bisulphite, but the cytidine residues of several C.G pairs at the ends of helical regions show some reactivity, and one of the proposed loops appears to contain six nucleotides, rather than the minimum of four suggested by the primary structure. Two cytidine residues that are thought to be 'looped out' by small helix imperfections also show some reactivity.

Base Sequence↗
PubMed1991

Effects of cytidine 5'-diphosphocholine administration on basal and growth hormone-releasing hormone-induced growth hormone secretion in elderly subjects.

The basal and GH-releasing hormone-stimulated secretion of GH declines in the elderly. We tested the ability of cytidine 5'-diphosphocholine, a drug used in the treatment of stroke and Parkinson's disease, to alter GH secretion in 11 healthy elderly volunteers, aged 69-84. Each subject received an iv infusion of 2 g of cytidine 5'-diphosphocholine or normal saline. GHRH and TRH were also administered during cytidine 5'-diphosphocholine infusions. The infusion of cytidine 5'-diphosphocholine induced a 4-fold (p less than 0.05) increase in serum GH levels over basal values. A small increase in GH was seen after GHRH administration. However, the addition of GHRH to the cytidine 5'-diphosphocholine infusion resulted in a GH response which was significantly greater than that seen after GHRH alone; the integrated concentration of GH was more than 2-fold greater in the cytidine 5'-diphosphocholine treated group (706.85 +/- 185.1 vs 248.9 +/- 61.4 micrograms.l-1.(120 min)-1; p = 0.01). The PRL and TSH responses to TRH were not significantly affected by cytidine 5'-diphosphocholine infusion, indicating that dopaminergic mechanisms are not involved. These studies demonstrate that cytidine 5'-diphosphocholine can enhance basal and GHRH-stimulated GH release in the elderly, but the mechanism of action of the drug remains unclear.

Aged↗
PubMed1991

Rho-dependent transcription termination. Characterization of the requirement for cytidine in the nascent transcript.

By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.

Bacteriophage lambda↗
PubMed2002

Antidepressant-like effects of cytidine in the forced swim test in rats.

BACKGROUND: Altered brain phospholipid metabolism may be involved in the pathophysiology of cocaine dependence and mood disorders. Evidence suggests that citicoline, a rate-limiting metabolite for phospholipid synthesis, reduces cocaine craving in human addicts. Because antidepressants can reduce cocaine craving, we explored in rats the possibility that citicoline has antidepressant effects. We also tested the primary metabolites of citicoline, cytidine and choline. METHODS: We examined if citicoline or metabolites alter immobility in the forced swim test. We used two scoring methods: latency to become immobile, a simple method that identifies antidepressants, and behavioral sampling, a complex method that differentiates antidepressants according to pharmacological mechanisms. RESULTS: Over a range of doses, citicoline did not affect behavior in the forced swim test. At molar equivalent doses, cytidine dramatically decreased immobility, whereas choline tended to increase immobility. The effects of cytidine resemble those of desipramine, a standard tricyclic antidepressant. None of the treatments affected locomotor activity, and cytidine did not establish conditioned place preferences. CONCLUSIONS: Citicoline does not have effects in the forced swim test, but its primary metabolites have opposing effects: cytidine has antidepressant-like actions, whereas choline has prodepressant-like actions. At antidepressant doses, cytidine lacks stimulant and rewarding properties. This is the first report of potential antidepressant effects of cytidine.

Animals↗
PubMed1999

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine.

Cytidine and choline, present in cytidine 5'-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100-500 microM) to [3H]-arachidonic acid (50 microM, 0.2 microCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [3H]-phosphatidylcholine (PtdCho), [3H]-phosphatidylinositol (PtdIno) and [3H]-phosphatidylethanolamine (PtdEtn) (by up to 27+/-3%, 16+/-3% and 11+/-3%, respectively, means+/-S.E.M.). This effect was seen with 8-18 h of incubation. The incorporation of [3H]-oleic acid into [3H]-PtdCho was even more enhanced (by up to 42+/-3%) as were the incorporations of [14C]-choline and [3H]-glycerol. The effects of choline and cytidine were enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [3H]-arachidonic acid into [3H]-PtdEtn, [3H]-PtdIno and [3H]-PtdCho. Arachidonic acid (10-200 microM) alone failed to affect the incorporation of [14C]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.

Animals↗
PubMed1998

Substrate connectivity effects in the transition state for cytidine deaminase.

The binding properties of substrates and competitive inhibitors of Escherichia coli cytidine deaminase are compared with those of the fragments obtained by cutting these ligands at several positions including the glycosidic bond. In contrast with the normal substrate cytidine (kcat/Km = 2.6 x 10(6) M-1 s-1), cytosine is found to serve as an extremely slow substrate (kcat/Km = 1.8 x 10(-3) M-1 s-1), despite the ability of cytosine to enter any active site that can accommodate the normal substrate cytidine. Spontaneous nonenzymatic deamination proceeds at similar rates for cytosine and cytidine at pH 7 and 25 degrees C, indicating that substituent ribose exerts little effect on the intrinsic reactivity of cytidine in solution. Dividing knon by kcat/Km, the maximal Kd value of the enzyme's complex with the altered substrate in the transition state is estimated as 6.1 x 10(-8) M for cytosine, very much higher than the value (1.2 x 10(-16) M) estimated for cytidine. The Kd value of ribofuranose, the missing substituent, is roughly 1.8 x 10(-2) M, as indicated by the Ki values of D-ribose and 1-methyl-D-ribofuranoside as competitive inhibitors. Thus, the free energy of binding of the altered substrate in the transition state is 9.5 kcal/mol more favorable for the whole molecule cytidine than for the sum of those of its parts, cytosine plus ribofuranose. As a separate molecule, however, ribose shows no detectable effect on the enzyme's activity on cytosine. Connectivity effects of similar magnitude are indicated by the equilibrium binding affinities of inhibitors. Thus, the Ki value of the transition state analogue inhibitor zebularine hydrate (1.2 x 10(-12) M) is very much lower than the combined affinities of N-ribofuranosylurea (1.6 x 10(-4) M) and allyl alcohol (0.14 M), indicating that the glycoside bond, by its presence, exerts a connectivity effect of 9.9 kcal/mol on the observed free energy of binding.

Binding Sites↗
PubMed1980

Uridine and cytidine metabolism following inhibition of de novo pyrimidine synthesis by pyrazofurin.

Pyrazofurin, an inhibitor of orotidylate decarboxylase, imposes an absolute nutritional requirement for exogenous uridine to maintain normal growth of L5178Y, P388, L1210, W256 and S180 cells in vitro. The amount of uridine necessary for cell division when de novo uridine nucleotide synthesis is inhibited by pyrazofurin is: L5178Y, 30.5; P388, 39.7; L1210, 53.3: W256, 70.6; and S180, 886 fmol/cell. Cytidine, which can be deaminated to uridine, will substitute for uridine to maintain normal cell growth in the presence of growth-inhibitory concentrations of pyrazofurin (5 microM). The requirements for cytidine and uridine are identical. If cytidine deamination is prevented by tetrahydrouridine (100 microM), cytidine can no longer support growth in the presence of pyrazofurin. Cytidine and uridine, as expected, are additive in their effect to permit normal growth of pyrazofurin treated cells. Tetrahydrouridine does not alter this additive effect, indicating that when both nucleotides are added to pyrazofurin treated cells each nucleotide replenishes their respective nucleotide pools and cytidine deamination is unnecessary to allow cell growth. Incorporation of [14C]uridine into the acid insoluble cell fraction of L5178Y cells was 25 fmol/cell at 48 h and remained constant during the remaining growth of the pyrazofurin treated cell suspension. The [14C]uridine acid soluble pool of 4 fmol/cell also was maximum at 48 h but declined during the subsequent growth of the suspension culture to approx. 2 fmol/cell at 96 h. This decline in the acid soluble pool is correlated with a 42% decrease in modal cell volume during this phase of cell growth which would maintain a constant specific activity of uridine in this pool. This may explain the decline in the acid soluble pool while the acid insoluble pool remains constant during growth of suspension cultures of L51878Y cells. The block in pyrimidine synthesis de novo induced by pyrazofurin provides a useful and quick method for the evaluation of uridine and cytidine metabolism of tumor cell specimens.

Amides↗
PubMed1992

Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (ccd) gene.

The structural gene that encodes cytidine deaminase (cdd) in Escherichia coli was cloned from Kohara phage lambda 365 (7F1), and its nucleotide sequence was determined. Plasmids harboring the gene complemented chromosomal cdd mutations, enhanced cytidine deaminase activity in cell extracts, and directed the synthesis of a protein identical in mass and N-terminal amino acid sequence with cytidine deaminase purified from wild-type bacteria. Metal analysis of the purified, plasmid-encoded deaminase indicated a single atom of tightly bound zinc per subunit. Earlier work has shown that bacterial cytidine deaminase and mammalian adenosine deaminase are remarkably alike in their mechanisms of action, in their free energies of interaction with analogue inhibitors resembling tetrahedral intermediates in nucleophilic substitution, and in their ability to discriminate between analogue inhibitors differing by a single hydroxyl group. In contrast to these shared catalytic similarities, the deduced amino acid sequence of E. coli cytidine deaminase (monomer MW 31,540) differs markedly from the mammalian adenosine deaminase sequence suggesting major differences in their tertiary structures. Nevertheless, cytidine deaminase and mammalian plus bacterial adenosine deaminases share a single region (TVHA) of sequence identity that is tentatively identified as part of the cytidine deaminase active site.

Amino Acid Sequence↗
PubMed1980

Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions.

The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.

Base Sequence↗
PubMed1998

Complementary truncations of a hydrogen bond to ribose involved in transition-state stabilization by cytidine deaminase.

The crystal structure of the complex formed between Escherichia coli cytidine deaminase and the transition-state analogue inhibitor 3,4-dihydrouridine [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., & Carter, C. W. (1994) J. Mol. Biol. 235, 635] shows the presence of an H-bond between Glu-91 and the 3'-OH group of substituent ribose, a part of the substrate that is not directly involved in its chemical transformation. To test the contribution of this interaction to transition-state stabilization, Glu-91 was converted to alanine. The mutant enzyme is very much less active than the wild-type enzyme, with a 500-fold increase in Km and a 32-fold reduction in kcat using cytidine as substrate. No change in secondary structure is evident in the circular dichroic spectrum. As measured by kcat/Km, Glu-91 thus appears to stabilize the transition state for cytidine deamination by an overall factor of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy. To test the contribution of this interaction in the opposite sense, the 3'-OH group of the substrate was replaced by a hydrogen atom. Comparing 3'-deoxycytidine with cytidine, the native enzyme shows a 17-fold increase in Km and a 400-fold decrease in kcat, indicating that the 3'-hydroxyl group of cytidine stabilizes the transition state for deamination by an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in free energy, as measured by kcat/Km. After one binding partner has been removed, however, the effect of removing the remaining partner is relatively slight. For the mutant enzyme E91A, removal of the 3'-hydroxyl group from substrate cytidine reduces kcat/Km by a factor of only 3. Complete removal of substituent ribose reduces the wild-type enzyme's kcat/Km by a factor of more than 10(8); thus, substituent ribose, although distant from the site of chemical transformation of the substrate, contributes at least 11 kcal to the free energy of stabilization of the transition state for cytidine deamination, matching the apparent contribution to transition state binding made by the 4-OH group of the pyrimidine ring, which is at the site of substrate transformation [Frick, L., Yang, C., Marquez, V. E., & Wolfenden, R. (1989) Biochemistry 28, 9423].

Amino Acid Substitution↗
PubMed1997

Allosteric mechanism of induction of CytR-regulated gene expression. Cytr repressor-cytidine interaction.

Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281 or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

Allosteric Regulation↗
PubMed2003

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools.

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

Adenosine Triphosphate↗
PubMed1982

Uptake of cytidine by isolated, perfused mouse liver.

Mouse livers were perfused at 22 degrees C with an oxygenated salts medium containing [5-3H]cytidine and [carboxyl-14C]inulin. The cellular uptake of cytidine was determined from the 3H content of liver samples less that present in the extracellular (inulin) space of the samples. Time courses of cytidine uptake were biphasic with initial phases which were approximately linear for 15 s and had time zero values that approximated the extracellular space. Rates of cytidine uptake derived from the initial phase of uptake evidently represented rates of membrane transport because (i) initial rates were saturable, and (ii) cytidine uptake was blocked by the nucleoside transport inhibitor, nitrobenzylthioinosine (NBMPR). Treatment of mice with the 5'-monophosphate of NBMPR (greater than 0.2 mg/kg, injected i.p.) 30-40 min prior to the perfusion also blocked cytidine entry into livers. An apparent half-saturation constant of about 10(-3) M and a maximum rate of about 1 mumol . g-1 . min-1 were estimated for the NBMPR-sensitive transport of cytidine into mouse liver cells.

Adenine Nucleotides↗
PubMedDate not supplied

[Double modification of cytidine residues in DNA].

The reaction of O-beta-diethylaminoethylhydroxylamine (O-beta-HA) with cytidine was studied and its mechanism was shown to be analogous to that of the reaction of hydroxylamine of O-methylhydroxylamine with cytidine. In experiments involving reaction of denatured DNA with O-beta-HA., Sephadex G-15 columns were used for the quantitative separation of normal and modified nucleosides after enzymatic hydrolysis of modified DNA by exonuclease A5 followed by alkaline phosphatase treatment. DNA cytidine residues of free cytidine with O-beta-HA. Modified cytidines can form complex with phosphotungstic acid (PTA). It was shown that one mole of PTA was bound per one mole of modified cytidine either in DNA or in free state. Electron microscopic examination of denatured DNA molecules modified by O-beta-HA and reacted with PTA revealed linear arrays of electron-scattering spots which presumably correspond to PTA molecules complexed with modified cytidine in DNA chains.

Alkaline Phosphatase↗