guanosine-triphosphate and Carcinoma--Ehrlich-Tumor

AMP deaminase has a key position in regulation of the pool of adenine nucleotides and energetics in malignant cells. The aim of this investigation was to elucidate mechanism of regulation of activity of AMP deaminase in intact tumor cells. Using tiazofurin, which profoundly decreases the level of GTP, and guanosine which markedly increases its concentration in the Ehrlich ascites tumor cells under aerobic conditions, we have shown that this nucleotide is probably the major regulator of AMP deaminase activity in situ. This regulation, however, should be considered in relation to the time and the changes which occur during transition of the cells from aerobic to anaerobic phase, and reverse, when other factors, like concentrations of the substrate and ATP are involved.

Topics: AMP Deaminase; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Guanosine; Guanosine Triphosphate; Liver Neoplasms, Experimental; Mice; Rats; Rats, Sprague-Dawley; Ribavirin; Tumor Cells, Cultured

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl- which might be consistent with a H2PO4-/Cl- exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K+ and Cl- channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na+ and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl- channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Topics: Adenine; Adenosine; Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Chloride Channels; Chlorides; CHO Cells; Cricetinae; Guanine; Guanosine; Guanosine Triphosphate; Kinetics; Mice; Potassium Channels; Potassium Chloride; Purines; Ribonucleotides; Tumor Cells, Cultured

The involvement of ribosomal RNA in the binding of eukaryotic elongation factor eEF-2 to the ribosome was investigated. eEF-2 was complexed to empty reassociated 80S ribosomes in the presence of the nonhydrolyzable GTP analogue GuoPP[CH2]P. The formed complex was treated with dimethyl sulfate, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and micrococcus nuclease to allow specific modification at single-stranded regions of the rRNAs. The sites of modification were localized by primer extension using complementary deoxynucleotide primers and reverse transcriptase. The modification pattern was compared to that obtained from 80S ribosomes lacking bound eEF-2. Binding of the factor to the ribosome resulted in the protection of specific sites in both 18S and 28S rRNA, while the reactivity of 5.8S rRNA was unchanged. In 18S rRNA, the affected nucleotides were localized to the 5'- and 3'-domains, and in 28S rRNA the protected nucleotides were seen in domains II, IV, and V. The alpha-sarcin/ricin loop in domain VI of 28S rRNA was inaccessible for chemical modification even in the absence of bound eEF-2. However, the bound factor protected A4256, located in the alpha-sarcin/ricin loop, from ricin-induced depurination.

Topics: Animals; Base Sequence; Binding Sites; Carcinoma, Ehrlich Tumor; CME-Carbodiimide; Cross-Linking Reagents; Guanosine Triphosphate; Mice; Micrococcal Nuclease; Molecular Sequence Data; Nucleic Acid Conformation; Peptide Elongation Factor 2; Peptide Elongation Factors; Ribosomes; RNA, Ribosomal, 18S; RNA, Ribosomal, 28S; Sulfuric Acid Esters

A particulate fraction consisting of heavy organelles such as nuclei and mitochondria was prepared from Ehrlich ascites tumor cells. From this fraction we have purified a GTP-binding protein with a molecular mass of 33 kDa (MTG33) by guanidine hydrochloride extraction followed by four steps of column chromatography. The Kd value of MTG33 for GTP was 17 nM. [alpha-32P]GTP-binding to MTG33 was inhibited by GTP and GDP, but not appreciably by ATP, CTP, UTP, or GMP. MTG33 hydrolyzed GTP to GDP at a rate of 4.5 mmol/min/mol protein. Subcellular fractionation analysis of mouse liver revealed that the heavy mitochondrial fraction contained the highest level of MTG33. Furthermore, dual immunofluorescence examination indicated that the staining of NIH 3T3 cells with anti-MTG33 antibody is coincident with the distribution of mitochondrial succinate dehydrogenase. Of the mouse organs examined, the heart contained the highest level of MTG33. These results strongly suggest that MTG33 is a GTP-binding protein located in mitochondria.

Topics: 3T3 Cells; Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Cell Nucleus; Fluorescent Antibody Technique; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Male; Mice; Mitochondria; Mitochondria, Liver; Molecular Weight; Subcellular Fractions

The effect of ADP-ribosylation on the function of eukaryotic elongation factor 2 (EF-2) was investigated by kinetic analysis of the EF-2-catalyzed hydrolysis of GTP in the presence of ribosomes and by direct determination of the affinity of the modified factor for the ribosome. Under conditions where the concentration of EF-2 was rate-limiting, the ADP-ribosylation reduced the maximum rate of GTP hydrolysis and the second order rate constant Kcat/Km by approximately 50%. A similar decrease in Kcat and Kcat/Km was observed when the concentration of ribosomes were kept rate-limiting. The affinity of EF-2 for the pretranslocation type of ribosomes was reduced by 2 orders of magnitude after ADP-ribosylation. No effect was observed in the interaction with the post-translocation type of ribosomes, the ribosomal conformation responsible for activation of the EF-2-dependent GTPase. We conclude that the ADP-ribosylation affects both the association of the modified factor with pretranslocation ribosomes and the hydrolytic capacity of the factor.

Topics: Adenosine Diphosphate Ribose; Animals; Carcinoma, Ehrlich Tumor; Diphtheria Toxin; Guanosine Triphosphate; Kinetics; Mice; NAD; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphoproteins; Ribosomes; Ricin

The uridylation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells was examined. This reaction required ATP or GTP, although these nucleotides were not incorporated into U6 RNA itself. ATP and GTP could be replaced by their nonhydrolyzable analogues ATP gamma S and GTP gamma S. Therefore, hydrolysis of ATP or GTP is not necessary for the uridylation of U6 RNA, indicating that these nucleotides are effectors of this reaction. By chromatographies of a nuclear extract of Ehrlich ascites tumor cells on phosphocellulose and DEAE-cellulose, U6 RNA could be separated from an enzyme adding a uridine residue(s) to this RNA.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleus; Guanosine Triphosphate; Hydrolysis; RNA, Neoplasm; RNA, Small Nuclear; Uridine; Uridine Triphosphate

The hydrolysis of [3H]phosphatidylinositol (PI) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) by cytosolic inositide phosphodiesterase (phospholipase C) from Ehrlich ascites tumour cells was determined. Cytosolic fractions were prepared from tumour cells that had been cultivated for two days at low serum level (2%) in the presence of 1-oleoyl-2-acetyl-sn-glycerol (OAG). Cytosols from unstimulated cells (2% serum without OAG) were used for comparison. Phospholipase C acting on PI and PIP2 was significantly inhibited in the cytosol of OAG-stimulated cells. The suppressed enzyme was activated by Ca2+ and also by the guanine nucleotide GTP in a concentration-dependent manner independently of calcium ions. In the presence of Ca2+, GTP exerted a synergistic stimulatory effect. In contrast, GTP and GTP gamma S showed no effect on the phospholipase C activity of unstimulated cells. It is suggested that the suppressed PI- and PIP2-specific enzyme activity can be modulated by its susceptibility to Ca2+ ions and GTP probably via the GTP-binding protein.

Topics: Animals; Calcium Chloride; Carcinoma, Ehrlich Tumor; Diglycerides; Enzyme Activation; Glycerides; Guanosine Triphosphate; Kinetics; Mice; Type C Phospholipases

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Peptide Initiation Factors; Proteins; RNA, Transfer, Met; Temperature

The physiological correlation between NDP-kinase and the enzyme-associated guanine nucleotide binding proteins (G1 and G2) has been studied in vitro. It was found that incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases with one of the nucleoside 5'-diphosphates (NDPs) in the presence of divalent cations (Mg2+ and Ca2+) results in the formation of nucleoside 5'-triphosphates (NTPs) within 40 sec even at low temperatures (below 4 degrees C) without strict base-specificity; and high-energy phosphates on the phosphoenzyme can transfer preferentially to GDP on the guanine nucleotide binding proteins (G1, G2 and r-p21 protein) in the presence of 0.25 mM Ca2+ or 1 mM Mg2+ even if any other NDPs are present in the reaction mixtures. These observations suggest that NDP-kinase may be responsible for the phosphate-transfer between GDP on the guanine nucleotide binding proteins and its phosphoenzyme.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cations, Divalent; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Nucleoside-Diphosphate Kinase; Phosphoproteins; Phosphotransferases; Proto-Oncogene Proteins; Ribonucleotides

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5'-diphosphates in the presence of 1 mM Mg2+ or 0.25 mM Ca2+ results in the rapid formation of nucleoside 5'-triphosphates without strict base specificity; GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v-rasH p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca2+; and [32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v-rasH p21 protein, even if any other nucleoside 5'-diphosphates are present in the reaction mixture. Although [32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg2+, but not in the presence of 0.25 mM Ca2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.

Topics: Adenosine Diphosphate; Animals; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mice; Nucleoside-Diphosphate Kinase; Nucleotides; Phosphates; Phosphotransferases

Ehrlich carcinoma and P388 leukemia cells were rendered resistant to 4-carbamoylimidazolium 5-olate (SM-108), and assessments were made of biochemical and pharmacological determinants for the sensitivity to SM-108 using both sensitive and resistant sublines. We observed that the treatment of cells with SM-108 in vitro caused a remarkable decrease in the intracellular guanosine 5'-triphosphate pool level in sensitive but not in resistant sublines. There was no difference in the ability to take up SM-108 between sensitive and resistant sublines, but the cellular conversion of SM-108 to its nucleotide, which is the putative active anabolite of SM-108, proceeded only in sensitive sublines. Enzymological studies revealed that the activity of adenine phosphoribosyltransferase (EC 2.4.2.7), which is believed to conjugate SM-108 with 5-phospho-alpha-D-ribose 1-diphosphate, was very low in the resistant sublines. These results strongly support our previous hypothesis that SM-108 is activated by adenine phosphoribosyltransferase to SM-108-nucleotide which then inhibits hypoxanthine-5'-monophosphate dehydrogenase (EC 1.2.1.14), a key enzyme for the de novo synthesis of guanosine 5'-monophosphate.

Topics: Adenine Phosphoribosyltransferase; Animals; Carcinoma, Ehrlich Tumor; Cell Line; Drug Resistance; Guanosine Triphosphate; Imidazoles; IMP Dehydrogenase; Leukemia P388; Mice; Neoplasms, Experimental

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.

Topics: Animals; Carcinoma, Ehrlich Tumor; Electrophoresis, Polyacrylamide Gel; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Molecular Weight; Peptide Initiation Factors; Phosphorylation; Protein Biosynthesis; Protein Kinases; Proteins; Ribosomes; RNA, Transfer, Amino Acyl; Solubility; Vaccinia virus; Viral Proteins

A species of DNA polymerase alpha that is active in the ATP(GTP)-dependent conversion of MVM parvovirus single-stranded linear DNA to the duplex replicative form has been purified 4300-fold from Ehrlich ascites mouse tumour cells. The single-stranded----replicative form activity is maintained throughout ammonium sulfate precipitation, DEAE-cellulose, phosphocellulose and hydroxyapatite column chromatography and glycerol gradient sedimentation. Polypeptides with Mr = 230 000, 220 000, 183 000, 157 000, 125 000, 70 000, 65 000, 62 000, 57 000, 53 000 and 48 000 copurify with the single-stranded----replicative form activity, which sediments at approx. 10 S. The Mr = 183 000, 157 000 and 125 000 polypeptides exhibit catalytic activity when assayed in situ following SDS-polyacrylamide gel electrophoresis. The 10 S form of DNA polymerase alpha is functionally distinguishable from an 8.4 S form of the enzyme obtained from the same cells on the basis of single-stranded----replicative form activity. The single-stranded----replicative form activity of the 10 S enzyme is stable at 22 degrees C for up to 3 h, but exhibits a half life of only 5 min at 45 degrees C.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; DNA Polymerase II; DNA Replication; DNA, Single-Stranded; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Mice; Minute Virus of Mice; Molecular Weight; Parvoviridae

Eukaryotic cell polypeptide chain initiation factor eIF-2 forms ternary complexes with GTP and initiator Met-tRNAf. These complexes can be destabilized in vitro by the addition of salt-washed 40S ribosomal subunits. Our evidence suggests that this destabilization is mediated by GDP generated by premature hydrolysis of the GTP molecule present in the ternary complex. With complexes formed by using a partially purified preparation of eIF-2 from Ehrlich ascites tumor cells, it is possible to reverse the 40S subunit induced inhibition by creating conditions which eliminate free GDP from the system. This reversal probably occurs due to exchange of GTP for the GDP bound to the initiation factor, in a reaction catalyzed by another factor present in the eIF-2 preparation. However, if the eIF-2 has previously been phosphorylated by the reticulocyte heme-controlled repressor, the 40S subunit induced inhibition cannot be reversed by elimination of free GDP. The instability of initiation complexes containing eIF-2, together with the impairment of guanine nucleotide exchange after phosphorylation of eIF-2 [Clemens, M.J., Pain, V.M., Wong, S.-T., & Henshaw, E. C. (1982) Nature (London) 296, 93-95], may be an important aspect of the mechanism of the inhibition of translation by the heme-controlled repressor.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Mice; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Phosphorylation; Proteins; Reticulocytes; Ribosomes; RNA, Transfer, Amino Acyl; RNA, Transfer, Met

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.

Topics: Animals; Carcinoma, Ehrlich Tumor; Eukaryotic Initiation Factor-2; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mice; Molecular Weight; Peptide Initiation Factors; Phosphorylation; Proteins; RNA, Transfer, Amino Acyl

Topics: Animals; Carbon-Nitrogen Ligases; Carcinoma, Ehrlich Tumor; Guanosine Triphosphate; Kinetics; Ligases; Magnesium; Mice; Substrate Specificity

To determine the subcellular distribution of initiation-factor eIF-2 activity, Ehrlich ascites-cell homogenates were fractionated to give (a) a rapidly sedimenting fraction, (b) a microsomal fraction and (c) post-microsomal supernatant. The first two fractions were washed in 0.5 m-KCl to render the associated protein-synthesis factor soluble. As much as 60% of the total recoverable eIF-2 was obtained from the rapidly sedimenting material.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Centrifugation, Density Gradient; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Peptide Initiation Factors; Protein Binding; Proteins; RNA, Transfer; RNA, Transfer, Amino Acyl; Subcellular Fractions

CTP synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) was purified 200-fold from Ehrlich ascites tumor cells with about 50% purity as judged by polyacrylamide gel electrophoresis. The molecular weight estimated to be 118 000 by gel filtration. The optimal pH was 8.6. 2-Mercaptoethanol was required for optimal activity and stabilization of the enzyme. Magnesium was essential for the reaction and other divalent cations were ineffective. Ammonia could replace glutamine as the amino donor. When other substrates were at saturating concentrations, Michaelis-Menten kinetics were observed yielding Km values for UTP, ATP and glutamine of 0.18, 0.8 and 0.13 mM, respectively. With ATP at subsaturating concentration, the double-reciprocal plot for UTP saturation was sinusoidal and the Hill plot showed an n value of 1.3. The double-reciprocal plot for ATP saturation, when UTP was at subsaturating concentration, departed from Michaelis-Menten kinetics with an n value of 1.4. These data suggest the existence of cooperative interactions between enzyme and substrates at subsaturating concentrations of ATP or UTP. GTP was not essential, but it acted as an activator on the glutamine reaction; optimal activation was observed at 1 mM GTP. The affinity for glutamine was not affected by GTP.

Topics: Amino Acids; Animals; Carbon-Nitrogen Ligases; Carcinoma, Ehrlich Tumor; Cations, Divalent; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Hydrogen-Ion Concentration; Kinetics; Ligases; Molecular Weight; Sulfhydryl Reagents

Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cations, Divalent; Cell Nucleolus; Cytidine Triphosphate; DNA-Directed RNA Polymerases; Guanosine Triphosphate; Hydrogen-Ion Concentration; Nucleotides; Rifamycins; RNA; RNA Polymerase I; Uridine Monophosphate; Uridine Triphosphate

The tRNA nucleotidyltransferase activity (3H-CMP incorporation into 3'-terminus of tRNApC) in cytoplasmic fractions of various types of cells such as Ehrlich ascites tumor cells, mouse liver and spleen cells, rat spleen, lymph node, and macrophages cells was found to be dependent on the concentrations of nucleoside 5'-triphosphates (ATP, GTP, UTP, dATP, dGTP, dCTP, and/or dTTP). The purified tRNA nucleotidyltransferase did not show such dependency. The dependency of the enzyme activity on nucleoside 5'triphosphates in the crude cytoplasmic fractions was possibly due to the presence of inhibitors which interfere with the repair system of defective 3'-termini of tRNA. Two kinds of inhibitors were distinguishable in the cytoplasmic fractions. One was unstable on heat treatment at 55 decrees C and showed ribonuclease activity for the tRNA 3'-terminus. The other which lacked ribonuclease activity was rather stable to the heat treatment and inhibited purified tRNA nucleotidyltransferase. The actions of both inhibitors were suppressed by nucleoside 5'-triphosphates.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cytoplasm; Deoxyribonucleotides; Guanosine Triphosphate; Liver; Lymph Nodes; Macrophages; Male; Mice; Nucleotides; Rats; Ribonucleases; RNA Nucleotidyltransferases; RNA, Transfer; Spleen; Temperature

When protein biosynthesis is inhibited by either cycloheximide of puromycine, the nucleolar RNA synthesis of Ehrlich ascites tumor cells decreases by approximately 70% within 1 h, while the removal of these protein synthesis inhibitors causes a rapid recovery of nucleolar RNA synthesis, largely within 1 h. A similar pattern of decrease and recovery of endogenous RNA polymerase activity in isolated nucleoli or in nuclei (in the presence of alpha-amanitin) may be demonstrated after addition and removal of these drugs. Analysis of the molecular species of RNA polymerase I on a phosphocellulose column indicates that only the IB form of the enzyme decreases in the nucleoli of drug-treated cells and recovers quickly after resumption of protein synthesis. The finding that the activity of the IB form enzyme remains unchanged in the whole nuclei indicates that during cessation of protein synthesis RNA polymerase IB is either released from the nucleoli into the extranucleolar compartment or becomes so loosely bound to the nucleoli that it is leached out from the nucleoli during their isolation. By using a system of assaying free, nucleolar-template bound and total RNA polymerase I activities, data supporting the above interpretation have been obtained. Namely, in isolated nuclei free enzyme activity increases with a concomitant decrease in bound enzyme activity during protein synthesis inhibition, while the total enzyme activity remains unchanged. In isolated nucleoli, both total and bound enzyme activities decreases on protein synthesis inhibition but recover quickly on its resumption. The putative bound enzyme, fractionated with the aid of actinomycin D, is exclusively IB form, whereas the unbound enzyme consists of both IA and IB forms as previously demonstrated (1). No conversion of IB form polymerase to IA form was noted on prolonged sonication in our system. The levels of ATP and GTP in the cell did not change appreciably either during cessation or resumption of protein synthesis in these cells. The data support the previous conclusion that some short-lived protein(s) is required to maintain the normal level of ribosomal RNA transcription (2) and further suggest that the protein is required to facilitate reinitiation of the transcription by RNA polymerase IB in the nucleolus.

Topics: Adenosine Triphosphate; Amanitins; Animals; Carcinoma, Ehrlich Tumor; Cell Nucleolus; Cell Nucleus; Cycloheximide; Dactinomycin; Guanosine Triphosphate; Liver; Mice; Protein Biosynthesis; Puromycin; Rats; RNA; RNA Polymerase I

Topics: Animals; Carcinoma, Ehrlich Tumor; Centrifugation, Density Gradient; Guanosine Triphosphate; Liver; Macromolecular Substances; Methionine; Mice; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Rabbits; Rats; Reticulocytes; Ribosomes; RNA, Transfer

Experiments were conducted to test the hypothesis that intracellular concentrations of guanosine triphosphate (GTP) regulate the activity and hormonal sensitivity of adenylate cyclase in intact cells. By appropriate treatments, the GTP concentrations of Ehrlich ascites tumor cells could be varied between 28% and 680% of control values. Cyclic AMP concentrations were measured before and after addition of epinephrine in cells containing this range of GTP concentratins. Basal cyclic AMP concentrations were unaffected by changes in GTP concentrations. In cells containing lowered concentrations of GTP, the cyclic AMP concentration following addition of epinephrine was half that in control cells. Elevation of GTP concentrations above normal and had no effect on cyclic AMP concentrations following epinephrine treatment.

Topics: Adenylyl Cyclases; Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Cyclic AMP; Cytosol; Epinephrine; Guanosine Triphosphate; Guanylyl Imidodiphosphate

Amino acid starvation of Ehrlich ascites cells leads to a significant decrease of the intracellular ATP concentration concomitant with a marked decrease in nucleolar RNA polymerase activity. Addition of 8-bromoguanosine 3':5'-monophosphate (br8cGMP) to the amino-acid-deficient culture medium increased the cellular ATP levels and restored the rRNA synthesis capacity of nucleoli to control levels. Exogenous br8cAMP overcame the effects of br8cGMP. Administration of br8cAMP to exponentially growing ascites cells resulted in a shrinkage of ATP levels and in an inhibition of nucleolar RNA synthesis similar to that observed under shift-down conditions. These effects of br8cAMP could be antagonized by exogenous br8cGMP or hypoxanthine. Since the br8cGMP-induced increase in the total adenine nucleotides was abolished in the presence of azaserine (an inhibitor of the amidation of formylglycineamide ribonucleotide) it is concluded that cyclic nucleotides exert at least a part of their regulatory effects on cell proliferation by regulating nucleotide biosynthesis de novo.

Topics: Adenosine Triphosphate; Animals; Azaserine; Carcinoma, Ehrlich Tumor; Cell Line; Cell Nucleolus; Guanosine Triphosphate; Nucleotides, Cyclic; Purine Nucleosides; RNA, Ribosomal; RNA, Transfer

One hundred and sixty-one purine analogs and derivatives were tested for their ability to inhibit ten parameters of purine metabolism in Ehrlich ascites tumor cells incubated in vitro with radioactive hypoxanthine. Sixty-seven compounds were inhibitory against at least one parameter and 30 were inhibitory against two or more.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cyclic GMP; Energy Metabolism; Glutamine; Guanine Nucleotides; Guanosine Triphosphate; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; In Vitro Techniques; Inosine Nucleotides; Ketone Oxidoreductases; Ligases; Lyases; Mice; Purines

The regulatory properties of adenylate deaminase (EC 3.5.4.6) from Ehrlich ascites tumor cells suggest that the reaction catalyzed by this enzyme serves to protect the cell against sharp decreases in the adenylate energy charge by removing adenosine 5'-monophosphate generated when the rate of utilization of adenosine triphosphate is suddenly increased. The enzyme is effectively inhibited under normal physiological conditions of high energy charge (0.9) and 4 to 5 mM adenine nucleotide pool size. The reaction is sharply activated by a decrease in the energy charge in the physiological range (0.9 to 0.6). At low energy charge (0.6), decrease in the size of the pool causes a marked and nonlinear decrease in the rate of the deaminase reaction. This effect presumably serves to prevent excessive depletion of the adenine nucleotide pool. Calculations based on the kinetic data obtained in this study show that the AMP deaminase reaction can account for the well-established alteration of adenine nucleotide metabolism that is observed following addition of glucose or 2-deoxyglucose to intact ascites cells.

Topics: Adenine Nucleotides; AMP Deaminase; Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Guanosine Triphosphate; Nucleotide Deaminases; Phosphates

Ehrlich ascites tumor cells containing radioactive ATP were incubated in vitro with a range of concentrations of 2-deoxyglucose in order to produce different rates of ATP catabolism. Concentrations of all radioactive products of ATP catabolism were measured, and apparent rates of adenylate deaminase and inosinate dehydrogenase and of adenylate and inosinate dephosphorylation were calculated. It was concluded that these processes were reggulated primarily by the rate of formation of substrate, and to a lesser extent in some cases, by substrate concentration. No evidence was obtained for regulation of these processes by the concentration of ATP. The deoxyglucose-induced catabolism of radioactive GTP was also studied. When ATP catabolism was induced by incubation with 2,4-dinitrophenol, time courses of accumulation of purine nucleoside monophosphates and rates of alternative pathways of their metabolism were quite different than when deoxyglucose was used.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Aminohydrolases; Animals; Antimetabolites; Carcinoma, Ehrlich Tumor; Deoxyglucose; Guanosine Triphosphate; Inosine Nucleotides; Kinetics; Mice; Phosphotransferases; Time Factors

Topics: Adenine; Adenosine Triphosphate; Amides; Animals; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Chromatography, Ion Exchange; Glycine; Guanine; Guanosine Triphosphate; Mice; Pentosephosphates; Pentosyltransferases; Phosphoric Acids; Purines; Ribose; Time Factors

Topics: Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Guanosine Triphosphate; Peptide Elongation Factors; Ribosomes; RNA, Transfer

Topics: Adenine; Animals; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Cells, Cultured; Chromatography, DEAE-Cellulose; Chromatography, Paper; Guanine; Guanosine; Guanosine Triphosphate; Hypoxanthines; Inosine; Leukemia L1210; Mice; Mycophenolic Acid; Nucleotides; Oxidoreductases; Purines; Spectrophotometry, Ultraviolet; Structure-Activity Relationship

Topics: Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Cell-Free System; Cells, Cultured; Guanine Nucleotides; Guanosine Triphosphate; Organophosphonates; Peptide Elongation Factors; Phenylalanine; Poly U; Protein Biosynthesis; Ribosomes; RNA, Transfer

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Animals; Arginine; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Cell-Free System; Centrifugation, Density Gradient; Guanosine Triphosphate; Hepatectomy; Liver; Liver Regeneration; Neoplasm Proteins; Puromycin; Rats; Ribonucleases; Ribosomes; RNA, Transfer; Time Factors

Topics: Adenosine Triphosphate; Agar; Animals; Carcinoma, Ehrlich Tumor; Cell Fractionation; Cell Nucleus; Cytosine Nucleotides; Deoxyribonucleases; DNA; Electrophoresis; Guanosine Triphosphate; Hot Temperature; Liver; Male; Methods; Mice; Phenols; Phosphorus Isotopes; Rats; RNA; RNA, Ribosomal; Uracil Nucleotides

Topics: Amino Acids; Animals; Anti-Bacterial Agents; Basidiomycota; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Cell Nucleus; DNA-Directed RNA Polymerases; Guanosine Triphosphate; Kinetics; Leucine; Mice; Mycotoxins; Neoplasm Proteins; Oligopeptides; Peptides, Cyclic; RNA, Neoplasm; Structure-Activity Relationship; Time Factors; Tritium

Topics: Adenine; Adenosine Triphosphate; Animals; Carbon Radioisotopes; Carcinoma, Ehrlich Tumor; Cells, Cultured; Chromatography, Thin Layer; Guanine; Guanosine Triphosphate; Hypoxanthines; Kinetics; Mice; Models, Biological; Purine Nucleotides

Cell-free extracts prepared from Ehrlich ascites and mouse L cells synthesize viral proteins in response to encephalomyocarditis virus, mouse Elberfeld virus, and mengovirus ribonucleic acid. Although HeLa cell extracts are inactive, their ribosomes are functional in the presence of heterologous supernatant fractions. Synthesis depends upon the addition of adenosine triphosphate, guanosine triphosphate, an energy-generating system, and 4 mm Mg(2+). Initiation is completed during the first 10 to 20 min of incubation, but chain elongation continues for 1 hr or more. The products are of higher molecular weight than virion structural proteins and resemble polypeptides formed in virus-infected cells during a short pulse. Tryptic peptides of virion proteins and in vitro products are similar for all three cardioviruses.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Line; Cell-Free System; Electrophoresis, Disc; Encephalomyocarditis virus; Genetic Code; Genetics, Microbial; Guanosine Triphosphate; HeLa Cells; Humans; L Cells; Magnesium; Mice; Molecular Weight; Peptide Biosynthesis; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Peptides; Ribosomes; RNA, Viral; Subcellular Fractions; Time Factors; Viral Proteins

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Antineoplastic Agents; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Guanine Nucleotides; Guanosine Triphosphate; Hypoxanthines; Inosine Nucleotides; Ligases; Mice; Mice, Inbred ICR; Oxidoreductases; Pentosyltransferases; Phosphotransferases; Purines; Succinates

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cells, Cultured; Guanosine Triphosphate; Hydrolysis; In Vitro Techniques; Mice; Nucleotides; Phosphates; Phosphorus Isotopes; Proteins; Serine; Threonine; Time Factors

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Cytosine Nucleotides; Deoxyribonucleotides; Drug Stability; Female; Guanosine Triphosphate; Hot Temperature; Hydrogen-Ion Concentration; Idoxuridine; Iodine; Kinetics; Magnesium; Mice; Temperature; Thymidine; Thymidine Kinase; Thymine Nucleotides; Uracil Nucleotides