Pyrimidine Nucleoside Monophosphate Kinase Research Articles

The Drosophila melanogaster UMP-CMP kinase cDNA encodes an N-terminal mitochondrial import signal

Drosophila melanogaster cells express a multi-substrate deoxyribonucleoside kinase that phosphorylates both purine and pyrimidine deoxyribonucleosides. The subsequent phosphorylation step is catalyzed by nucleoside monophosphate kinases. We have cloned and characterized the D. melanogaster UMP-CMP kinase ( Dm.UMP-CMP kinase) to further study the nucleotide metabolizing enzymes in these cells. The kinase, encoded by the dak1 gene, was ≈60% similar to the human UMP-CMP kinase and predominantly phosphorylated CMP, dCMP, and UMP. Expression analysis showed that the Dm.UMP-CMP kinase mRNA was constitutively expressed throughout the Drosophila development. The open-reading frame of the Dm.UMP-CMP kinase cDNA was extended in the 5 ′-region compared to UMP-CMP kinases of other species. The extended region encoded an N-terminal sequence with properties characteristic of a mitochondrial import signal. Expression of the enzyme in fusion with the green fluorescent protein verified that the N-terminal signal targeted the enzyme to mitochondria. This is the first time a mitochondrial pyrimidine nucleoside monophosphate kinase has been cloned from any organism and we discuss the implication of this finding for deoxyribonucleoside salvage in both Drosophila and other organisms.

Pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase deficiency.

Decreased Pyrimidine Nucleoside Monophosphate Kinase Activity in Sickle Erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin- sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5′-nucleotidase (P5N), 6- phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin- sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Metabolism of 1-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC) in Human Embryonic Lung Cells

The acyclic nucleotide analogue HPMPC is in human embryonic lung cells, cultured in vitro, transformed to its mono- and diphosphates (HPMPCp and HPMPCpp) and HPMPCp-choline; the synthesis of HPMPCp is catalysed by pyrimidine nucleoside monophosphate kinase (EC 2.7.4.14); HPMPCp-choline (analogue of CDP-choline) is formed from HPMPCpp and choline phosphate in the presence of CTP:phosphorylcholine cytidylyltransferase (EC 2.7.7.15). These metabolites persist for a long time in the cellular pool even after HPMPC has been removed from the medium; on the other hand, they efflux to the medium. Neither HPMPC, nor any of its metabolites interfere with intracellular CDP-choline level.

Enzymatic synthesis of cytidine 5′-diphosphate using pyrimidine nucleoside monophosphate kinase

Nucleoside monophosphate kinase (NMPK, EC 2.7.4.4, from bovine liver or yeast) has been used to prepare cytidine 5′-diphosphate (CDP). The enzyme catalyses the reversible ( K eq ≅ 1) reaction of a pyrimidine nucleoside 5′-monophosphate and ATP to produce a nucleoside 5′-diphosphate and ADP. Equilibrium mixtures of CMP, CDP, ADP, and ATP were obtained from the reaction of CMP, ATP, and magnesium chloride with NMPK. The soluble enzyme could be recovered and reused but an enzyme activity half-life of only ca. 2 days was observed. Stabilization of enzyme activity by immobilization via covalent bonding to a carrier surface, and by gel entrapment, was examined. Immobilization yields were optimized by varying protein loading, pH, temperature, ionic strength, type of buffer, and concentration of enzyme substrates. The highest yields of immobilized NMPK activity were obtained by gel entrapment in a poly(acrylamide-co- N-acryloxysuccinimide) gel crosslinked with triethylenetetramine (PAN-500); NMPK immobilized using this method exhibited increased stability of enzyme activity compared to the unimmobilized enzyme.

Pyrimidine nucleoside monophosphate kinase hyperactivity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The pyrimidine nucleoside triphosphates (CTP, UTP) increase in the pyrimidine 5'-nucleotidase (P5N) deficient red blood cell (RBC) to a greater degree than do the pyrimidine nucleoside monophosphates (CMP, UMP). Pyrimidine nucleoside monophosphate (PNMP) kinase phosphorylates CMP and UMP to their respective phosphodiesters. We tested the hypothesis that increased PNMP kinase activity contributes to the disproportionate increase in CTP and UTP in the P5N deficient RBC. CMP and UMP kinase activities were increased in high reticulocyte (4.4 +/- 2.1 and 8.5 +/- 3.3 mumol/ml RBC per minute) compared to normal RBC (2.8 +/- 1.0 and 6.0 +/- 2.5 mumol/ml RBC per minute). P5N deficient RBC (n = 2) had significantly increased CMP and UMP kinase activities (14.0 and 26.5 mumol/ml RBC per minute). UMP and CDP-ethanolamine were able to increase the activity of CMP kinase in crude haemolysate and the activity of partially purified enzyme. Since the Km for CMP of CMP kinase was 33 mumol/l in P5N deficient RBC and since the CMP concentration is 25-90 mumol/l in the P5N deficient RBC, the enzyme should be nearly saturated with CMP in the P5N deficient RBC. Thus, PNMP kinase hyperactivity appears to contribute to the disproportionate increase in CTP and UTP in the P5N deficient RBC.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: A kinetic analysis of the reaction mechanism

A kinetic analysis of the reaction mechanism of pyrimidine nucleoside monophosphate kinase was carried out with a highly purified enzyme preparation from rat bone marrow cells. The results of initial rate and product inhibition studies provided insight into the mode of action of the enzyme. The data support the views that (i) the reaction mechanism is sequential and nonequilibrium in nature, (ii) Substrates bind to the enzyme in a random order, (iii) Substrate binding is cooperative. That is, the binding of the first substrate facilitates the binding of the second substrate, (iv) UMP can bind to the purine site on the enzyme, resulting in substrate inhibition, (v) Product inhibition can result from the binding of UDP to either the pyrimidine or purine site, or from the binding of ADP to the purine site.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: Chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 °C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 m m dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: Purification to high specific activity by a two-step affinity chromatography procedure

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (>600 μmol/min/mg protein), and was obtained with 30–36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.

Purification and properties of pyrimidine nucleoside monophosphate kinase from baker's yeast.

Pyrimidine nucleoside monophosphate kinase was purified from baker's yeast to 280-fold. The molecular weight of the enzyme was calculated to be 26, 000 by gel filtration. With ATP as a phosphate donor, UMP was the most active phosphate acceptor. Besides UMP, the kinase also phosphorylated CMP, dCMP and dUMP. Km values for UMP, CMP, dCMP and dUMP were 0.052, 0.071, 0.54 and 8.5mM, respectively. The kinase was activated by preincubation with dithiothreitol. The extent of the activation depended on the concentration of dithiothreitol and incubation time. HgCl2, p-CMB and N-ethylmaleimide inhibited this enzyme.

Kinetics and equilibria of pyrimidine nucleoside monophosphate kinase from human erythrocytes

The common type of pyrimidine nucleoside monophosphate kinase (ATP:CMP phosphotransferase, EC 2.7.4.14), purified 50 000-fold from human erythrocytes, reacted with a wide variety of nucleotides, but only ATP, dATP, UMP and CMP were good substrates. The optimum Mg 2+ concentration, 2–3 mM, was generally independent of substrate concentration, of the nature of the substrate, and of the direction of the reaction. Kinetic studies indicated that a ternary complex was formed, that the substrates were bound at two unlike sites, and that the order of addition of subtrates was random. Equilibrium constants were ATP + UMP 0.98, ATP + CMP 1.59, dATP + UMP 1.13, and ATP + AMP 1.20.

Synthesis of sugar-modified nucleoside 5′-triphosphates with partially purified nucleotide kinases from calf thymus

The partial purification of some nucleoside monophosphate kinases (ATP:nucleosidemonophosphate phosphotransferases, EC 2.7.4.4) from calf thymus by chromatography on Blue Sepharose to remove interfering phosphatase activity is described. Their specificities towards nucleoside monophosphates modified in the sugar are investigated. Pyrimidine nucleoside monophosphate kinase is not very much affected by such modifications, whereas GMP kinase does not tolerate such alteration. The effect on AMP kinase is intermediate.

A rapid procedure for separation and purification of purine and pyrimidine nucleoside monophosphate kinases

1. 1. The partial purification of pyrimidine nucleoside monophosphate kinase, adenylate kinase and guanylate kinase has been applied directly to cytosol preparations of rat liver, erythrocytes, Novikoff hepatoma, mouse adenocarcinoma and human liver. 2. 2. Solutions of dCTP or CTP (1.0 mM) appear to be the most efficient eluents of pyrimidine nucleoside monophosphate kinase and erythrocyte adenylate kinase from cellulose phosphate column. 3. 3. Liver and hepatoma adenylate kinase require ATP for elution. 4. 4. The erythrocyte adenylate kinase is inhibited by pHMB ( K i 0.0087 mM) and NEM ( K i 0.0611 mM) and has a low affinity for AMP ( K m 0.717 mM) in contrast to the liver adenylate kinase which is not inhibited by NEM and pHMB and has a higher affinity for AMP ( K m 0.0951 mM).

Human erythrocyte pyrimidine nucleoside monophosphate kinase. Partial purification and properties of two allelic gene products.

Human pyrimidine nucleoside monophosphate kinase is a polymorphic enzyme having two allelic gene products, UMPK 1 and UMPK 2, in several populations. A procedure is described for the partial purification of this enzyme from human red blood cells resulting in a 1500-fold purification of the enzyme for UMPK 1 and 583-fold for UMPK 2. The purified enzyme preparation catalyzed the phosphorylation of UMP, CMP, and dCMP, and used ATP as the preferred phosphate donor. The heavy metals, mercury, and copper, were found to be strong inhibitors of pyrimidine nucleoside monophosphate kinase activity. EDTA was found to protect the enzyme from inactivation by the heavy metals, and 2-mercaptoethanol stabilized the enzyme during purification. UMPK 1 and UMPK 2 were found to have similar kinetic properties; however, UMPK 2 had a slower electrophoretic mobility and greater thermolability than UMPK 1.

Activation of rat liver pyrimidine nucleoside monophosphate kinase

The activity of the pyrimidine nucleoside monophosphate kinase (ATP:dCMP phosphotransferase, EC 2.7.4.14) from rat liver is dependent upon the presence of sulhydryl-reducing agents. Addition to the inactive enzyme of 2-mercaptoethanol (5 mM), a reagent specific for cleavage of disulfide bonds, effects a reduction in molecular weight from approx. 53 000 to 17 000, measured by molecular sieve chromatography. This low molecular weight form is partially active in the presence of 2-mercaptoethanol (5 mM). In absence of 2-mercaptoethanol, the low molecular weight form is inactive. Higher concentrations of 2-mercaptoethanol (50 mM) fully reactivate the CMP(ATP) kinase activity followed by dCMP(ATP) and CMP(dCTP) kinase activities in a sequential manner, without further change in molecular weight. Alkylation by iodoacetamide of the enzyme at different stages of reactivation in dithiothreitol suggests an ordered appearance of the various enzyme activities. Furthermore, iodoacetamide inactivates the fully active enzyme. Thioredoxin was found to activate the enzyme in a manner similar to 2-mercaptoethanol and dithiothreitol. These results are consistent with the interpretation that the mechanism of activation of the enzyme involves cleavage of inter- and intramolecular disulfide bonds.

A pyrimidine nucleoside monophosphate kinase from rat liver.

A pyrimidine nucleoside monophosphate kinase has been purified 2100-fold from rat liver. With ATP and dATP as phosphate donors the kinase uses CMP, dCMP, and UMP as phosphate acceptors. Ara-CMP is also phosphorylated by the kinase. In contrast to dCMP and UMP, CMP can be phosphorylated by dCTP. CTP and ara-CTP cannot substitute for dCTP. The stringent specificity of the phosphate donor site for ATP and dATP is lost when CMP serves as acceptor. All nucleoside triphosphates act as donors to a significant extent. No evidence has been found to suggest more than one enzyme. All activities, to different degrees, are strictly dependent upon preincubation at 37 degrees with a sulfhydryl reducing agent. Various reagents (85 mM) are ranked in order of increasing effectiveness of reactivation as follows: dithiothretiol greater than glutathione larger than or equal to 2-mercaptoethanol greater than L-cysteine greater than DL-alpha-lipoic acid. A NADP+-dependent thioredoxin (17 muM)-thioredoxin reductase system from Novikoff ascites rat tumor was found to be the most powerful reducing agent tested. CTP, dCTP, UTP, and dTTP (1 mM) do not affect the kinase activity regardless of the phosphate acceptor.

Localization in Escherichia coli B of two enzymatic sites of action by 1-formylisoquinoline thiosemicarbazone (IQ-1) on ribonucleic acid biosynthetic pathways

The site(s) of action of 1-formylisoquinoline thiosemicarbazone (IQ-1) on RNA biosynthetic pathways was investigated in Escherichia coli B by measuring the effects of this drug on the conversion of 3H-uridine and 3H-adenosine to acid-soluble nucleotide forms. The incorporation of 3H-uridine into acid-soluble UMP and CMP was unaffected by a 50 per cent growth-inhibitory concentration of IQ-1; however, the progression of radioactivity from pyrimidine nucleoside monophosphates to di- and triphosphates was markedly depressed. In contrast, the conversion of 3H-adenosine to purine nucleotide forms was unaffected by IQ-1. A partially purified preparation of ATP: nucleoside monophosphate phosphotransferase from E. coli was used to show that pyrimidine nucleoside monophosphate kinase activity was sensitive to IQ-1. A second locus of action appeared to be RNA polymerase; variable inhibition, which seemed to depend upon the degree of purity of the enzyme, was produced by IQ-1. Several structurally related compounds were tested for potency against pyrimidine nucleoside monophosphate kinase and RNA polymerase; the results showed close correlation between the potential for inhibition of growth and the degree of interference with the activity of these enzymes, suggesting the involvement of these lesions in the bacteriostatic action of IQ-1.