Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase

Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.     

A CMP-N-acetylneuraminic acid synthetase purified from a marine bacterium, Photobacterium leiognathi JT-SHIZ-145

A cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg(2+) for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.     

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens.

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe Clostridium perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40 degrees C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent Km values for cytidine 5'-diphosphate-diacylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).     

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.     

Repression of cytidine triphosphate synthetase in Salmonella typhimurium by pyrimidines during uridine nucleotide depletion

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.     

Visualization of nucleolar substructure in cultured human fibroblasts by magnesium-activated adenosine triphosphatase reaction

Discrete sites of adenosine triphosphatase (ATPase) activity were demonstrated within the nucleoli of unfixed cultured human fibroblasts (IMR90, VA13, and AG2804 cells) by an adaptation, for electron microscopic cyto-chemistry, of Wachstein and Meisel's lead nitrate method. The majority of nucleoli contained more than one ATPase-positive region, but the total ATPase-positive material appeared to occupy only a minor portion of the nucleolar volume. These regions were roughly spherical with an irregular contour, and at times appeared to be components of perinucleolar chromatin or to be located adjacent to nucleolar interstices. The distribution of these regions within the nucleolus and their segregation by actinomycin D suggested that the ATPase-positive regions correspond to the fibrillar centers, which represent nucleolar organizer regions. The cytochemically demonstrable nucleolar ATPase was strictly dependent on the presence of divalent cations. Optimal reactions was seen at 5 mM Mg2+, but near optimal activity was obtained with lower concentrations of Mg2+ in the presence of Ca2+. Calcium alone and Mn2+ alone produced suboptimal reaction. Studies with different nucleoside phosphates as reaction substrates showed that the enzyme is specific for adenosine derivatives, ATP and dATP being equally good substrates. Guanosine triphosphate, cytidine triphosphate, uridine triphosphate, and d-thymidine triphosphate were ineffective as substrates, as were nucleoside mono- and diphosphates and other phosphate esters tested. It is suggested that the cytochemical ATPase reaction visualized the regions of the nucleolus in which ribosomal DNA of intranucleolar chromatin is undergoing conformational alterations.     

Regulation of rat-liver glycoprotein: N-acetylneuraminic acid transferase activity by pyrimidine nucleotides.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N-acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N-Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide-sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.     

Cytidylate cyclase activity in mouse tissues: the enzymatic conversion of cytidine 5'-triphosphate to cytidine 3',5'-cyclic monophosphate (cyclic CMP)

Cytidylate cyclase activity, which enzymatically converts cytidine 5'-triphosphate (CTP) to cytidine 3',5'-cyclic monophosphate (cyclic CMP), has been demonstrated in mouse tissue homogenates by use of a highly sensitive enzyme immunoassay (EIA) specific for cyclic CMP. Cyclic CMP formation is dependent on the amount of homogenate and on the incubation time. Although the enzyme activity was detected at wide ranges of pH from 6.8 to 11.5, the maximal activity was observed at around pH 9.4. The optimal temperature was 37 degrees C. Cytidylate cyclase activity was almost completely lost if the homogenates were heated at 90 degrees C for 3 min prior to use. The enzyme reaction exhibited typical Michaelis-Menten kinetics with an apparent Km for CTP of approx. 0.31 mM. Cyclic CMP formation was greatly enhanced with 4 mM Mn2+, Mg2+, Co2+; Mn2+ was the most effective. Fe2+ and Ca2+ were without effect. Cu2+ and Zn2+ at a concentration of 0.1 to 0.5 mM were inhibitory to Mn2+-dependent activity. Moreover, the enzyme activity was inhibited by several nucleotides including ATP, ADP, 5'-AMP, and GTP. Cytidylate cyclase activity was found to be present in all homogenates from a variety of mouse tissues examined except heart, with the highest level found in brain, and the lowest in liver.     

Nucleotide Phosphohydrolase in Purified Vaccinia Virus

Purified infectious vaccinia virus has been shown to contain an enzyme or enzymes that remove the terminal phosphate group from adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). The K(m) for ATP of this enzyme is 5.5 x 10(-4)m, and the relative rates of the reaction with ATP, GTP, UTP, and CTP are 1.00, 0.34, 0.15, and 0.29, respectively. The virus enzyme does not react with any of the diphosphates. The rate of the reaction is proportional to the amount of virus added and is linear for 130 min. The virus nucleotide phosphohydrolase activity is greatly stimulated by Mg(++) and very slightly stimulated by Ca(++). The small residual activity observed in the absence of divalent cations is completely inhibited by ethylenediaminetetraacetic acid. Neither Na(+) nor K(+) ions, nor any mixture of these, was found to stimulate the reaction significantly, and ouabain, at 10(-4)m, inhibited the reaction by only 27%. The response of the vaccinia enzyme to mono- and divalent cations and to ouabain indicates that the vaccinia enzyme has different properties from those associated with microsomes and mitochondria.     

Cytidine triphosphate: phosphatidic acid cytidyltransferase in Escherichia coli

An enzyme has been found in particulate fractions of Escherichia coli that catalyzes the incorporation of cytidine triphosphate (CTP) into lipid in the presence of exogenous phosphatidic acid and Mg(++). The product has been identified enzymatically and by chromatography as cytidine diphosphate diglyceride. The reaction is optimal at a pH of 6.5 and Mg(++) concentration of 5-10 mm. The apparent K(m) for CTP is 7 x 10(-4)M and for phosphatidic acid, 2 x 10(-3)M. The reaction rate falls off rapidly with time and ceases entirely after 1 hr as the result of inactivation of the system by Mg(++).     

Studies on acylneuraminate cytidylytransferase from human placenta

The presence of acylneuraminate cytidylyltransferase has not been shown in human placenta so far. In this paper, evidence is put forward to demonstrate the presence of this enzyme in human placenta. The soluble enzyme was partially purified 30-fold using ammonium sulphate fractionation (30-60%) and DEAE Sephadex A-50 chromatography. This enzyme preparation had a specific activity of 0.75 mkat/kg protein. The pH optimum of the reaction was 9.0. The Km values for N-acetylneuraminic acid and cytidine triphosphate were 2.2 mM and 4 mM respectively. It was also found that the presence of magnesium is necessary for the enzyme activity.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

Human thymidylate kinase. Purification, characterization, and kinetic behavior of the thymidylate kinase derived from chronic myelocytic leukemia.

Thymidylate kinase derived from the blast cells of human chronic myelocytic leukemia was purified 2186-fold to near homogeneity by means of alcohol precipitation, alumina-Cgamma gel fractionation, calcium phosphate gel fraction, ultrafiltration, and affinity column chromatography. The molecular weight was estimated by glycerol gradient centrifugation to be 50,000. This enzyme had an optimal activity at pH 7.1 and required a divalent cation in order to catalyze the reaction. Mg2+ and Mn2+ were found to be the preferential divalent cations. The activation energy was estimated to be 19.1 kcal/mol at pH 7.2. Initial velocity study suggested that the reaction followed a sequential mechanism. Mg2+ ATP had a Km of 0.25 mM and dTMP had a Km of 40 micrometer. The enzyme was unstable even at 4 degrees. In the presence of ATP or dTMP the enzyme maintained its activity. Purine triphosphate nucleosides were found to be better phosphate donors than the pyrimidine triphosphate nucleosides. ATP and dATP had a lower Km and a higher Vmax than GTP and dGTP. dTMP was the only preferred phosphate receptor among all the monophosphate nucleotides tested dTTP and IdUTP competed with both substrates and inhibited the reaction with a Ki of 0.75 mM and 1.1 mM, respectively.     

Properties of a membrane-bound cardiolipin synthetase from Lactobacillus plantarum.

Cardiolipin (CL) synthetase of Lactobacillus plantarum 17-5 catalyzed the stoichiometric conversion of 2 mol of phosphatidylglycerol to 1 mol of CL. The enzyme activity was linear with time for 30 min at 37 C and with protein concentration between 20 and 200 mug of protein per ml. The enzyme was membrane associated, had a pH optimum of 5.1 in phosphate buffer, and was not stimulated by Mg2+, and the activity was not affected by the addition of ethylenediaminetetraacetic acid, cytidine diphosphate diglyceride, or cytidine triphosphate. The reaction was inhibited about 95% by Triton X-100 (0.5% final concentration) and by CL, the end product of the reaction. The activity of this enzyme was studied as a function of growth. The CL synthetase specific activity was highest during the early and midexponential growth phases, as was the cellular content of CL. The results demonstrate a correlation between enzyme-specific activity and lipid content of the cells.     

Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia.

A new fast assay procedure for increasing deoxyuridine triphosphate nucleotidohydrolase activity was developed. With this assay procedure, this enzyme derived from blast cells of patients with acute lymphocytic leukemia was purified at least 1218-fold. The molecular weight was estimated by gel filtration to be 43,000. The enzyme exhibited optimal activity over a pH range of 7 to 8 and the activation energy was estimated to be 6.5 kcal/mol at pH 7.5. While the enzyme had activity in the absence of added divalent cations, the activity could be inhibited by EDTA but not by phenanthroline. The inhibition caused by EDTA could be reversed by Mg2+ or Zn2+. The enzyme had maximal activity in the presence of Mg2+ (40 muM) and Mg2+ (4 mM) stabilized the enzyme at 37 degrees C. Cupric ion (0.5 mM) inhibited (50%) enzyme activity in the presence or absence of Mg2+. The substrate for the enzyme was dUTP and the apparent Km was 1 muM. No other deoxyribonucleoside or ribonucleoside triphosphate served as a substrate for the enzyme.     

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     

A potent nucleoside triphosphate hydrolase from the parasitic protozoan Toxoplasma gondii. Purification, some properties, and activation by thiol compounds

A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.     

Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.     

Reverse and forward reactions of carbamyl phosphokinase from Streptococcus faecalis R. Participation of nucleotides and reaction mechanisms

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies of approx. 1 mM free Mg2+ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg2+ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.     

Cyclopentenylcytosine triphosphate. Formation and inhibition of CTP synthetase

Cyclopentenylcytosine (CPEC) is phosphorylated in L1210 cells with CPEC triphosphate as the major metabolite. Partially purified uridine-cytidine kinase catalyzes the initial phosphorylation of cyclopentenylcytosine with an apparent Km of 196 +/- 9 microM, and cyclopentenylcytosine is a competitive inhibitor of cytidine phosphorylation by this enzyme with a Ki value of 144 +/- 14 microM. Examination of the CTP synthetase activity in extracts of L1210 cells revealed a dose-dependent decrease on exposure of cells to CPEC. Synthesis of CPEC triphosphate by an enzymatic method permitted direct examination of the inhibition of partially purified CTP synthetase. CPEC triphosphate inhibited bovine CTP synthetase with a median inhibitory concentration of 6 microM, whereas CPEC mono- and diphosphates were ineffective. CTP synthetase showed a classical Michaelis-Menten hyperbolic plot of velocity and UTP concentration in the presence of saturating concentrations of ATP and glutamine, but CPEC triphosphate induced sigmoidal kinetic plots. The Hill coefficient was calculated to be 3.2.