Nucleoside diphosphate kinase activity in purified cores of vesicular stomatitis virus.

Purified cores of vesicular stomatitis virus contain an enzymatic activity that converts GDP, UDP, and CDP into their corresponding triphosphates using ATP as the phosphate donor. Thus, the virion-associated RNA polymerase can synthesize mRNA normally in vitro even when one of the ribonucleoside triphosphates is replaced by its corresponding diphosphate. RNA synthesis does not proceed if ATP is replaced by ADP. Similarly RNA synthesis is impaired if CDP and UDP are present in the same reaction. The role of the nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6) in vesicular stomatitis virus mRNA synthesis in vitro is discussed.     

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.     

Nucleoside diphosphate kinase A as a controller of AMP-kinase in airway epithelia

This review integrates recent understanding of a novel role for NDPK-A in two related directions: Firstly, its role in an airway epithelial cell when bound to the luminal (apical) membrane and secondly in the cytosol of many different cells (epithelial and non-epithelial) where an isoform-specific interaction occurs with a regulatory partner, AMPKα1. Thus NDPK-A is present in both a membrane and cytosolic environment but in the apical membrane, its roles are not understood in detail; preliminary data suggest that it co-localises with the cystic fibrosis protein (CFTR). In cytosol, we find that NDPK-A is coupled to the catalytic alpha1 isoform of the AMP-activated protein kinase (AMPKα subunit), which is part of a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. We find that ATP is located within this complex and ‘fed’ from NDPK to AMPK without ever ‘seeing’ bulk solution. Importantly, the reverse can also happen such that AMPK activity can be made to decline when NDPK-A ‘steals’ ATP from AMPK. Thus we propose a novel paradigm in NDPK-A function by suggesting that AMP-kinase can be regulated by NDPK-A, independently of AMP.     

Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling

The results of numerous investigations during the last 20 years show that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its β-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to GDP in the active center of G protein α-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.     

Nucleoside diphosphate kinase and the flagellar movement of glycerinated spermatozoa.

Nucleoside diphosphate kinase [EC 2.7.4.6.] of sperm flagella and Tetrahymena cilia is detected mostly in the outer fiber fraction, suggesting an association of the enzyme with the outer fiber microtubules. The enzyme does not catalyze transphosphorylation between microtubule-bound GDP and exogenous ATP. Even when undulatory movement of glycerinated sperm is induced by MgATP, no phosphorylation is detected in the outer fiber fraction. These facts do not appear to support the hypothesis that the phosphorylation of microtubule-bound GDP is involved in the mechanism of flagellar movement.     

Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.

Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule-associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co-purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP.     

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.     

Nucleoside diphosphate kinase of Mycobacterium tuberculosis acts as GTPase-activating protein for Rho-GTPases

Several bacterial pathogens secrete proteins into the host cells that act as GTPase-activating proteins (GAPs) for Rho-GTPases and convert GTP-bound active form to GDP-bound inactive form. However, no such effector molecule has been identified in Mycobacterium tuberculosis. In this study, we show that culture supernatant of M. tuberculosis H(37)Rv harbors a protein that stimulates the conversion of GTP-bound Rho-GTPases to the GDP-bound form. Nucleoside diphosphate kinase (Ndk) was identified as this culture supernatant protein that stimulated in vitro GTP hydrolysis by members of Rho-GTPases. The histidine-117 mutant of Ndk, which is impaired for autophosphorylation and nucleotide-binding activities, shows GAP activity. These results suggest that Ndk of M. tuberculosis functions as a Rho-GAP to downregulate Rho-GTPases, and this activity may aid in pathogenesis of the bacteria.     

Nucleoside diphosphate kinase: role in bacterial growth, virulence, cell signalling and polysaccharide synthesis

Nucleoside diphosphate kinase (Ndk) is an important enzyme that generates nucleoside triphosphates (NTPs) or their deoxy derivatives by terminal phosphotransfer from an NTP such as ATP or GTP to any nucleoside diphosphate or its deoxy derivative. As NTPs, particularly GTP, are important for cellular macromolecular synthesis and signalling mechanisms, Ndk plays an important role in bacterial growth, signal transduction and pathogenicity. Specific examples of the role of Ndk in regulating growth, NTP formation and cell surface polysaccharide synthesis in two respiratory tract pathogens, Pseudomonas aeruginosa and Mycobacterium tuberculosis , are discussed.     

Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.     

Nucleoside diphosphate kinase from Myxococcus xanthus. I. Cloning and sequencing of the gene

By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction. The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe. The DNA sequence of the gene was determined, which did not show similarity with other known proteins. The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein. Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization. This result indicates that this gene is essential for cell growth. In the following paper (Mu?oz-Dorado, J., Inouye, S., and Inouye, M. (1990) J. Biol. Chem. 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.     

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.     

Apparent ATP-linked succinate thiokinase activity and its relation to nucleoside diphosphate kinase in mitochondrial matrix preparations from rabbit.

The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool.     

Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.     

Nucleoside diphosphate kinase at the cell surface of neoplastic human cells in culture

Strong evidence is given that a nucleoside diphosphate kinase is present to some extent on the surface of intact neoplastic cells in culture. Experiments could be performed with cells cultured in a few plates to which an incubation medium was added. The cells were firmly attached to the supporting medium and remained viable during the incubation procedure. Determinations of lactate dehydrogenase were carried out to rule out any possible contamination from the culturing medium as well as from the cell interior. From these analyses, a procedure was developed which easily removed the last traces of the culturing medium and which showed that there was no leakage of intracellular lactate dehydrogenase during the incubation procedure. There was a rather insignificant diffusion of nucleoside diphosphate kinase into the incubation medium. In common with other nucleoside diphosphate kinases, the glioma cell surface enzyme seemed to be nonspecific with regard to nucleotide substrates.     

A possible role of protein kinase C in signal-induced lysosomal enzyme release

In platelets, activation of protein kinase C and mobilization of Ca2+ were selectively induced by the addition of 1-oleoyl-2-acetyl-glycerol and a low concentration of A23187, respectively (Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704). Using this procedure evidence was obtained suggesting that the protein phosphorylation and Ca2+ mobilization were both essential and synergistically effective to cause release of lysosomal acid hydrolases such as N-acetylglucosaminidase. A similar observation was made for the lysosomal enzyme release from rat neutrophils.     

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2an6 algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction