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PubMed1995

Synthetic lipopeptides activate nucleoside diphosphate kinase in HL-60 membranes.

We have put forward the hypothesis that lipopeptides (LPs) activate GTP hydrolysis by Gi-proteins in HL-60 membranes via activation of nucleoside diphosphate kinase (NDPK) as does mastoparan (MP). Therefore, we compared the effects of LPs and MP on NDPK- and GTPase activation in HL-60 membranes. In native membranes, LPs effectively activated GTP hydrolysis and moderately activated GTP formation. In solubilized membranes, the effect of LPs on GTP formation was enhanced whereas the one on GTP hydrolysis was abolished. The NDPK substrate GDP enhanced the relative stimulatory effect of LPs and MP on GTP hydrolysis in HL-60 membranes in the absence of a NTP-regenerating system. A NTP-regenerating system abrogated the potentiating effect of GDP on MP-action, whereas the effect on LP-stimulated GTP-hydrolysis was enhanced. Our data show that LPs activate NDPK in HL-60 membranes and that this activation may account for their G-protein-stimulatory activity. Membrane solubilization may impair the transfer of GTP from NDPK to Gi-protein alpha-subunits and subsequent GTP hydrolysis, whereas GTP formation remains intact, augmenting the effect of LPs on the kinase. Finally, LP- and MP-induced NDPK activation may involve different pools of GDP.

Amino Acid Sequence↗
PubMed2000

The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells.

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.

Animals↗
PubMed1994

Refined X-ray structure of Dictyostelium discoideum nucleoside diphosphate kinase at 1.8 A resolution.

The X-ray structure of the nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been refined at 1.8 A resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 A. The Dictyostelium structure is described in detail and compared to those of Drosophila and Myxococcus xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular alpha/beta domain. The four-stranded beta-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve alpha-helices and a 20-residue segment called the Kpn loop. The beta-sheet is regular except for a beta-bulge in edge strand beta 2 and a gamma-turn at residue Ile120 just preceding strand beta 4. The latter may induce strain in the main chain near the active site His122. The alpha 1 beta 2 motif participates in forming dimers within the hexamer, helices alpha 1 and alpha 3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyostelium are conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococcus xanthus enzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.

Amino Acid Sequence↗
PubMed1995

The crystal structure of a human nucleoside diphosphate kinase, NM23-H2.

The 2.8 A resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myc oncogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthus NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is identical to the fold of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Amino Acid Sequence↗
PubMed1995

The gene for nucleoside diphosphate kinase functions as a mutator gene in Escherichia coli.

Nucleoside diphosphate (NDP) kinase is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in various cellular activities such as developmental control, signal transduction and metastasis in eukaryotic systems. In this study, the gene for NDP kinase of Escherichia coli (ndk) was disrupted and surprisingly found to be dispensable without any discernible effects on cell growth or morphology. However, a mutator phenotype was found in ndk-disruption strains; frequencies of spontaneous mutations to rifampicin resistance and nalidixic acid resistant significantly increased. A higher frequency in reversion mutations was observed with use of an amber mutation in the kanamycin-resistance gene in an ndk-disruption strain. Imbalance in dNTP pools, in particular a significant increase of the dCTP content was observed, which is likely to result in the higher spontaneous mutation rates. These results suggest that NDP kinase, although not essential, plays an important role in the appropriate balance of intracellular dNTP pools to maintain a high DNA replication fidelity. Strains with ndk- pykA- pykF- as well as ndk- scs- were constructed without any discernible effect on cell growth, indicating that there is yet another enzyme(s) catalyzing nucleoside triphosphate synthesis, in addition to NDP kinase, pyruvate kinases and succinyl CoA synthetase.

DNA Replication↗
PubMed1995

Endothelium-dependent relaxation in response to adenosine diphosphate is impaired under poor runoff conditions in the canine femoral artery.

To examine whether poor distal runoff conditions of the canine femoral artery modulates endothelium-dependent relaxations, we developed a poor distal runoff limb in the canine right femoral artery (poor runoff group). The left femoral artery was used as a control. Endothelium-dependent responses were examined in vitro. The rings of femoral artery were obtained from dogs pretreated with 6 weeks of poor distal runoff. In the control, flow rate and tau-variation were 79.1 ml/min and 214.2 dynes/cm2, respectively. In the poor runoff group, the flow rate and tau-variation were 27.4 ml/min and 52.7 dynes/cm2. There were significant differences between the two groups. In the rings taken from the control group contracted with norepinephrine, adenosine diphosphate (ADP) and A23187 caused endothelium-dependent relaxations. In the poor runoff group, the endothelium-dependent relaxations in response to ADP were impaired, while A23187 caused comparable endothelium-dependent relaxations. Direct relaxations in response to sodium nitroprusside were comparable between the two groups. These experiments indicate that under poor runoff conditions, the endothelium-dependent relaxations to ADP are impaired without changing the properties of the smooth muscle cells. This dysfunction of the endothelium under conditions of abnormal flow may accelerate a loss of late patency of the reconstructed arteries.

Adenosine Diphosphate↗
PubMed2001

Inexpensive purification of P450 reductase and other proteins using 2',5'-adenosine diphosphate agarose affinity columns.

Two reductases, P450 oxidoreductase and P450Bm-3 reductase, were purified on a 2',5'-adenosine diphosphate solid support. Although the efficiency of these columns is well established, the cost of the resin and the eluting material 2'-adenosine can be prohibitive. Herein we show that the less costly 2',3'-adenosine monophosphate is an excellent eluting material.

Adenosine Diphosphate↗
PubMed1994

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding.

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.

Amino Acid Sequence↗
PubMed1984

Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli.

An RNA-DNA hybridization assay was used to quantitate the ribonucleoside diphosphate reductase mRNA synthesis (nrd mRNA) to show that gene expression was dependent on protein synthesis. The increased nrd mRNA synthesis induced by inhibition of DNA synthesis was eliminated by simultaneous inhibition of protein synthesis. It was further found that protein synthesis is required not only initially but continuously during DNA inhibition for increased expression of nrd mRNA synthesis.

DNA Replication↗
PubMed1992

Role of nitric oxide in modulating permeability of hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin.

The goal of this study was to determine the role of the synthesis and release of nitric oxide in modulating alterations in microvascular permeability of the hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. We used intra-vital fluorescent microscopy to examine the permeability of the hamster cheek pouch to agonists before and following application of enzymatic inhibitors of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 0.01, 0.1, and 1.0 microM) and NW-nitro-L-arginine methyl ester (L-NAME; 0.01, 0.1, and 1.0 microM). Increases in permeability of the hamster cheek pouch were quantitated by the formation of microvascular leaky sites. ADP and bradykinin produced an increase in the number of venular leaky sites, and superfusion of L-NMMA and L-NAME significantly decreased ADP- and bradykinin-induced increases in microvascular permeability. To determine the specificity of nitric oxide blockade on microvascular permeability, we examined changes in permeability in response to adenosine, and examined the effects of D-NMMA on microvascular permeability. Adenosine-induced increases in permeability were not altered by treatment with L-NMMA, and D-NMMA did not inhibit ADP-induced increases in microvascular permeability. Thus, these findings suggest that production of nitric oxide, in response to application of ADP and bradykinin, has a role in modulating macromolecular permeability of the hamster cheek pouch in vivo.

Adenosine Diphosphate↗
PubMed1984

Diphosphate concentration does not correlate with the level of inorganic diphosphatase in Escherichia coli.

Partial inhibition of DNA synthesis stimulates the production of inorganic diphosphatase in Escherichia coli but the changes in diphosphate (PPi) level observed did not correlate with the enzyme activity. An accumulation of PPi was observed in the presence of inhibitors of RNA synthesis or nucleotide synthesis. In the former case the level of the enzyme did not change but in the latter case it increased. Thus the amount of inorganic diphosphatase alone does not determine the concentration of PPi in E. coli.

DNA Replication↗
PubMed2001

Nucleoside diphosphate kinase--a component of the [Na(+)]- and [Cl(-)]-sensitive phosphorylation cascade in human and murine airway epithelium.

We have shown that proteins within apically enriched fractions of human nasal respiratory epithelium vary their phosphohistidine content with ambient [Cl(-)] and other anion concentrations. This membrane-delimited phosphorylation cascade includes a multifunctional protein histidine kinase - nucleoside diphosphate kinase (NDPK). NDPK is itself a cascade component in both human and ovine airway, the self-phosphorylation of which is inhibited selectively by [Na(+)] in the presence of ATP (but not GTP). These findings led us to propose the existence of a dual anion-/cation-controlled phosphorylation-based "sensor" bound to the apical membrane. The present study showed that this cascade uses ATP to phosphorylate a group of proteins above 45 kDa (p45-group, identities unknown). Additionally, the Cl(-) dependence of ATP (but not GTP) phosphorylation is conditional on phosphatase activity and that interactions exist between the ATP- and GTP-phosphorylated components of the cascade under Cl(-)-free conditions. As a prelude to studies in cystic fibrosis (CF) mice, we showed in the present study that NDPK is present and functionally active in normal murine airway. Since NDPK is essential for UTP synthesis and regulates fetal gut development, G proteins, K(+) channels, neutrophil-mediated inflammation and pancreatic secretion, the presence of ion-regulated NDPK protein in mouse airway epithelium might aid understanding of the pathogenesis of CF.

Adenosine Triphosphate↗
PubMed1997

Effects of chronic administration of cytidine-5'-diphosphate choline (CDP choline) on the aged female mouse Harderian gland.

The effect of chronic intake of cytidine-5'-diphosphate choline (CDP choline), a precursor of the membrane lipid phosphatidylcholine, was analyzed in secretory cells of the Harderian gland (HG) of aged (26-month-old) female Swiss mice. These mice were treated with the drug from 12 months of age until they were 26 months old. The results obtained were compared with those of an age-matched control group and another adult (12-month-old) control group. In both control groups, the HGs displayed secretory tubules formed by a single squamous epithelium of lipid-secreting cells. The tubule lumens were occupied by numerous small pleomorphic porphyrin accretions. In contrast, the tubules of the CDP-choline-treated animals consisted of a single columnar epithelium, with lipid-secreting cells filled with lipid vacuoles. The most striking feature in the HGs of the CDP-choline group was the absence of prophyrin deposits. These observations indicate that porphyrin biosynthesis in female Swiss mice is maintained even in very old animals, and that chronic administration of CDP choline prevents porphyrin deposition in the HG of aged animals.

Aging↗
PubMed1998

Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli.

The E. coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. We found that the IciA protein binds specifically to the AT-rich upstream region of nrd promoter. In vivo overexpression of IciA increases the expression of nrd gene by four- to five-fold, suggesting that IciA functions as a transcriptional activator for the nrd gene.

Bacterial Proteins↗
PubMed2006

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase.

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.

Bacteriophage T4↗
PubMed2006

The nucleoside diphosphate kinase from mimivirus: a peculiar affinity for deoxypyrimidine nucleotides.

The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK(apm) exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDK(apm) revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDK(apm) to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDK(apm) might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDK(apm) over a recent acquisition from a eukaryotic organism by horizontal gene transfer.

Acanthamoeba↗
PubMed1987

Synthesis and structure determination of [8(-13)C]guanosine 5'-diphosphate.

A combined chemical and enzymatic synthesis of [8(-13)C]guanosine 5'-diphosphate (GDP) from H13COOH is described. About 35 mg nucleotide was obtained from 500 mg H13COOH. Analysis of the [8(-13)C]GDP by negative ion fast atom bombardment mass spectrometry and by 13C NMR confirmed that one atom of 13C was incorporated at the 8-position of the guanine ring at 90 +/- 10% enrichment. The chemical shift of the C(8) was 140.2 ppm downfield from internal trimethylsilylpropionate at neutral pH and room temperature, with a spin-spin coupling 1J(13C(8)-H(8] of 217 Hz and a 3J(13C(8)-H(1'] of 3.9 Hz.

Carbon Isotopes↗