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Aspergillus fumigatus is a major pathogen of invasive pulmonary aspergillosis with high mortality rate. The nucleoside diphosphate kinase of A. fumigatus, AfNDK (also called SwoH) is essential for its viability, however, its structural characteristic was unknown. In this study, we solved the crystal structure of AfNDK and found that it exists predominantly in form of tetramer in solution. Oligomeric form rather than dimeric form was essential for its kinase activity. The Arg30 and the C terminal amino acids were crucial for dimer-dimer interaction and the viability of A. fumigatus. Mutation V83F might make the secondary structure α5 helix protrude outward so that the whole protein structure became unstable at higher temperature, which might subsequently result to the inviability of A. fumigatus under 44 °C. In conclusion, the crystal structure of AfNDK was for the first time analyzed and the stability of the tetrameric form with dimer-dimer interaction were crucial for its function in A. fumigatus.

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.

Chondroitin sulfate proteoglycans (CSPGs) are a major component of glial scars, inhibiting axonal growth in the central nervous system. Protein tyrosine phosphatase, receptor type S (PTPσ) has been identified as a receptor for CSPGs, whereas its downstream signaling pathway remains to be fully understood. Here, we report that nucleoside diphosphate kinase 2 (NME2) interacts with PTPσ. We screened proteins associated with PTPσ by mass spectrometry, and obtained NME2. Immunoprecipitation analysis revealed that NME2 associated with the PTPσ intracellular domain in HEK-293T cells. NME2 was expressed in the cytoplasm and nucleus of cortical neurons, and knockdown of NME2 in the cortical neurons completely rescued neurite outgrowth inhibition induced by CSPGs. These results demonstrate that NME2 associates with PTPσ to elicit neurite outgrowth inhibition in response to CSPGs.

International audience; In stolon of white clover (Trifolium repens L), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS-PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS-PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress. (C) 2013 Elsevier Inc. All rights reserved.

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva’s apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.

Nucleoside diphosphate kinases (NDPKs) are conserved throughout evolution and have been shown to be involved in various biological phenomena. By functional screening in yeast, we identified a new member of the NDPK family, nm23-M5, which encodes a 211-amino acid protein with 86% identity to the human homolog Nm23-H5. Northern blot analysis revealed that nm23-M5 encodes two transcripts of 0.8 and 0.7 kb, which are highly and specifically expressed in adult testis. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that nm23-M5 transcripts first appear in pachytene spermatocytes and increase in abundance in subsequent stages. However, a low level of nm23-M5 mRNA was detected by RT-PCR in other tissues, such as ovary, brain, heart, and kidney. In situ hybridization studies showed that testicular nm23-M5 transcripts are localized in stage 12 to stage 16 spermatids in the neighboring lumen of seminiferous tubules. This distribution contrasts with that of Nm23-H5 transcripts, which are specifically found in spermatogonia and early spermatocytes. The heterologous expression of nm23-M5 in yeast cells confers protection from cell death induced by Bax, which is due to the generation of reactive oxygen species. Furthermore, overexpression of nm23-M5 in fibroblasts altered the cellular levels of several antioxidant enzymes, particularly glutathione peroxidase 5. Thus, we believe that the murine nm23-M5 gene plays an important role in late spermiogenesis by elevating the ability of late-stage spermatids to eliminate reactive oxygen species.

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.

Nucleoside diphosphate kinase (NDPK) was purified from Drosophila melanogaster by a combination of anion-exchange, hydroxyapatite, and reversed-phase chromatography. The identity of the purified enzyme was confirmed by sequencing internal peptides (the N-terminus appeared to be blocked). Post-translational modifications were investigated by using protein chemical and mass spectrometric methods. Analysis by nanoelectrospray ionization-mass spectrometry revealed that the mass of the enzyme was considerably smaller than that predicted from its amino acid sequence. Although its open-reading frame predicts a 153-residue polypeptide, the mature enzyme was found to comprise 152 amino acids, being modified by proteolytic removal of the initiator Met and N-acetylation of Ala2. This explains why the observed pI of the Drosophila enzyme is more acidic than that predicted from its amino acid sequence. No additional post-translational modifications such as glycosylation or O-phosphorylation, which have been identified on homologous NDPKs from other organisms, were detected on the Drosophila enzyme.

Although the gene responsible for multiple endocrine neoplasia type 1 (MEN1) has been identified, the function of its gene product, menin, is unknown. To examine the biological role of the MEN1 gene, we searched for associated proteins with a yeast two-hybrid system using the MEN1 cDNA fragment as bait. On screening a rat fetal brain embryonic day 17 library, in which a high level of MEN1 expression was detected, we identified a putative tumor metastasis suppressor nm23/nucleside diphosphate (NDP) kinase as an associated protein. This finding was confirmed by in vitro interaction assays based on glutathione S-transferase pull down experiments. The association required almost the entire menin protein, and several missense MEN1 mutations reported in MEN1 patients caused a loss of the binding activity for nm23. This result suggests that this interaction may play important roles in the biological functions of the menin protein, including tumor suppressor activity.

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is

Telomeres, the ends of eukaryotic chromosomes, are essential structures formed by specific protein-DNA complexes that protect chromosomes from degradation and end-to-end fusion. TRF1, a double-stranded telomeric TTAGGG-repeat binding protein, is associated with mammalian telomeres and controls telomere length by inhibiting the action of telomerase. We identified human nucleoside diphosphate kinase nm23-H2 as a human TRF1-interacting protein by yeast two-hybrid screening. In vitro-binding assays using different recombinant nucleoside diphosphate kinases showed that TRF1 predominantly binds the nm23-H2 isoform rather than nm23-H1. Electrophoretic mobility shift analysis revealed that the recombinant nm23-H2 protein can bind the single-stranded telomeric TTAGGG-repeat while it cannot bind the double-stranded telomeric repeat. The synthetic 20 base oligoribonucleotide, which consists of the template sequence CUAACCCUAAC and the adjacent region of the RNA component of human telomerase, was also found to form the complex with the recombinant nm23-H2 protein. Furthermore, the affinity of telomerase for its substrate was increased in vitro by presence of the plentiful nm23-H2 protein. These findings indicate a close relationship between nm23-H2 and human telomeres and suggest a new biological role for nucleoside diphosphate kinase.

The transcription of human nm23 genes (nm23-H1, nm23-H2) is involved in suppression of tumor metastasis or tumor progression. Therefore the characterization of transcriptional regulatory mechanisms for both nm23 genes is very important. In this study we have isolated and analyzed the 5′-flanking region of the human nm23-H2 gene and estimated the distance to 4 kb between nm23-H2 and nm23-H1 genes. We localized the known microsatellite D17S396 within this region. Futhermore the identification of possible binding sites for MYC proteins and additionally the NM23-H2 protein itself (the transcription factor PuF for c-myc gene activation) is of importance with respect to possible para- and autoregulatory interactions. A comparison of the promoter sequences of both human nm23 genes revealed no significant sequence homology.

Elongation of nascent polypeptides in a Dictyostelium discoideum in vitro translation system did not require the addition of ATP and GTP when creatine phosphate and creatine phosphokinase were present. However, depletion of the exogenous energy supply completely abolished incorporation of amino acids. Addition of dTTP, a nucleoside triphosphate that can be utilized by nucleoside diphosphate kinase (NDP kinase) to phosphorylate endogenous ADP and GDP, partially restored protein synthesis. Dictyostelium ribosomes were found to contain NDP kinase activity that could not be released by 1 M KCl. Thermal denaturation studies, specific inhibition with antibodies, and Western blotting identify the activity as cytosolic NDP kinase. These data support the idea that GTP can be fed into the translation machinery efficiently by NDP kinase associated with active ribosomes.

Proliferation of the androgen-dependent human prostate LNCaP cells was increased by the androgen DHT. Changes in the steady state level of the nm23 and c-myc mRNA in LNCaP cells, with or without 10 nM DHT, showed the nm23 mRNA to change rapidly, beginning to rise after 2 h and reaching its peak by 4 h of DHT treatment. In contrast, increases in the c-myc gene only became apparent after 4 h of treatment. Maximal increase of nm23 mRNA was observed at 10(-9) M DHT. The basal expression of c-myc and nm23 mRNAs was between 30-70% lower in the LNCaP cells, as compared with the androgen-independent PC-3 and JCA-1 human prostatic human carcinoma cells. Thus nm23 may be classified as a member of the early androgen-responsive genes.

Udgivelsesdato: 1994-Mar-15 We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK's from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

Expression of the nm23/NDP-K gene correlates with reduced metastasis in some tumors and with increased proliferation in both nontransformed and transformed cells in culture. Decreased nm23/NDP-K expression results in mitotic arrest in neuroblasts of developing Drosophila. In order to better understand the biological role(s) of nm23 in non-transformed cells, an nm23-specific antibody was introduced into rat embryo fibroblasts and effects on DNA synthesis and cell cycle progression were analyzed. Microinjection of the nm23 antibody inhibited cell division with no apparent effect on DNA synthesis. Control experiments revealed that the survival of cells injected with the nm23 antibody was similar to that of control antibody injected cells in the absence of cell division. These results suggest that in mammalian fibroblasts, as in Drosophila, nm23 expression may be necessary for progression through the cell cycle.

When the Gs in rat liver membranes was prelabeled with [32P]NAD and cholera toxin, solubilized with octylglucoside, and then analyzed by sucrose density gradient centrifugation, it was fractionated into two peaks with approximate molecular sizes of 12-13S and 3-4S. Pretreatment without or with GDP beta S of the labeled membranes resulted in a larger peak in the high molecular weight region, whereas pretreatment with glucagon plus GTP gamma S caused almost equal peaks in both regions. The affinity-purified anti-nucleoside diphosphate (NDP) kinase antibodies only precipitated the Gs in high molecular weight region. Under the same condition, small but significant NDP kinase activity was associated with the high molecular weight Gs region although a large portion of the enzyme activity was recovered in fractions where it alone should appear (6.2S). Both Lubrol-PX and digitonin solubilized the Gs in forms insensitive to immunoprecipitation by anti-NDP kinase antibodies although the latter detergent was able to solubilize the Gs in a high molecular weight form, that is, a ternary glucagon-receptor-G protein complex. These results demonstrate that Gs and membrane-associated NDP kinase may exist in part in a complexed form in membranes. Physiological relevance of the complex formation in membrane signal transduction is discussed.

AtNDPK2 is involved in transcriptional regulation in response to pathogen and abiotic stresses. AtNDPK2 -expressing transgenic rice plants showed regulation of the marker genes for chilling and oxidative stresses. In the present study, we produced AtNDPK2 -overexpressing transgenic rice lines using the co-transformation method. Morphologically, the transgenic plants, compared with the control plants, were growth retarded. We investigated how AtNDPK2 overexpression influences the response of rice plants to marker genes related to chilling and ROS stress. The accumulation of transcripts of pBC442 and pBC601 , related to chilling stress, was induced in AtNDPK2 -overexpressed rice plants. On further investigation, we found that OsAPX1 -, OsAPX2 -, and OsSodB -scavenging free-oxygen radicals, such as superoxide ( O 2 - ) and hydrogen peroxide (H 2 O 2 ), could be induced in AtNDPK2 -overexpressed rice plants. In particular, transcripts encoding pathogenesis-related (PR) proteins OsPR2 and OsPR4, as well as oxidative stress response proteins, were confirmed to change the gene expression in the transgenic rice plants. Together, these results suggest that AtNDPK2 plays a regulatory role in chilling and antioxidant signaling in plants.

Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47 kDa molecular weight. We isolated overlapping cDNAs of Lbc from human lung tissue. Full-length Lbc cDNA encodes 309 kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure. In order to analyze the regulatory mechanism of its activity, we searched for binding proteins. By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain. nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity. nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc. Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc. Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product.

In order to identify Nm23-H1’s structural motifs influencing its metastasis-inhibitory activity, we transfected DU 145 human prostate carcinoma cells with the expression vector encoding the Nm23-H1 protein with mutations at the following amino acids: serine-44, a phosphorylation site; proline-96, a site corresponding to the k-pn mutation that causes developmental defects in Drosophila ; and serine-120, a site of mutation in human neuroblastoma and phosphorylation. Significant decrease in colonization in soft agar and invasiveness of DU 145 cells was observed in the wild type nm23-H1 transfectants, and also in the serine-44 and serine-120 to alanine mutant nm23-H1 -transfected cell lines. However, the k-pn type proline-96 to serine (P96S) and neuroblastoma type serine-120 to glycine (S120G) mutations of Nm23-H1 abrogated its inhibitory activity on colonization and invasion. Meanwhile, all of the recombinant mutant Nm23-H1 proteins produced in Escherichia coli exhibited NDP kinase activity levels at the wild type protein, although the P96S and S120G mutant proteins exhibited decreased histidine protein kinase activity and autophosphorylation level, respectively. Interestingly, only two of the mutant recombinant Nm23-H1 proteins examined, P96S and S120G, exhibited reduced hexameric and increased dimeric oligomerization relative to the wild type. These correlative data suggest that the metastasis-suppressing activity of Nm23-H1 may depend on its oligomeric structure, but not on its NDP kinase activity.

Nucleoside diphosphate (NDP) kinase is an enzyme that transfers the gamma-phosphate of nucleoside triphosphates to nucleoside diphosphates. Besides this well-defined role, recent evidence suggests that NDP kinase may be implicated in a wide variety of essential cellular processes. In this paper, we showed that the NDP kinase of Drosophila exhibited protein kinase activity as well as autophosphorylation. Ovalbumin was phosphorylated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) or ATP. Protein kinase activity was not detected in NDP kinase mutant, abnormal wing discs (awd). These results suggest that this activity could be one of the functions of NDP kinase essential for normal fly development, since awd gene is lethal.

The putative metastasis suppressor genes, NME1(nm23-1) and NME2(nm23-2), were examined in a model system we developed to approximate the dissemination of melanoma from a primary skin tumor. We utilized two autologous human melanoma cell lines, IV Cl 1 and IV Cl 3, which displayed qualitatively different metastatic phenotypes following subdermal inoculation into nude mice. Highly metastatic IV Cl 1 cells expressed approximately 5 fold lower levels of protein encoded by NME genes than non-metastatic IV Cl 3 cells. Similar differences in NME protein levels were observed in tumors induced by the two cell lines in nude mice. There were no differences in NME mRNA levels between these two cell lines, suggesting that expression of these proteins is regulated at a post-transcriptional level. We found a ser122-pro mutation in the NME2 gene of metastatic IV Cl 1 cells. A similar ser120-gly mutation in NME1 has been found in human neuroblastoma, suggesting that mutation in this region may be a general phenomenon related to tumor progression. These mutations may have functional consequences since they eliminate potential phosphorylation sites and may affect the tertiary structure of mature protein complexes.

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.

Embryonal carcinoma in the human male is a pluripotential germ cell tumor (GCT), which is suggested to further differentiate to teratoma which displays somatic differentiation representing all three germinal layers. In a panel of 37 GCTs we determined frequency of loss of heterozygosity (LOH) and the level of expression of nucleoside diphosphate kinase (NDPK) genes NME 1 and NME 2. The frequency of LOH in teratomas (86%) was found to be highly significant (P < 0.01) compared to embryonal carcinomas (17%). We also found that the NME encoded proteins are expressed at a 4-5 fold lower level in teratomas compared to embryonal carcinomas. These findings lead us to hypothesize that a critical level of NDPK may be necessary for suppression of aberrant somatic differentiation.

Nucleoside diphosphate kinase has been shown to play a role in proliferation and development. Microtubules have been evoked as a possible target of NDP kinase action; in particular it was proposed that NDP kinase could regulate the cellular pool of polymerizable GTP-tubulin by direct phosphorylation of tubulin bound GDP. We show that this reaction does not occur in vitro and also that NDP kinase does not bind to microtubules both in the presence and absence of MAPs. Thus, any possible physiological effect of NDP kinase on microtubule dynamics is exerted only by modulating the concentrations of free guanine nucleotides in the vicinity of microtubules.

Mouse myeloid leukemic line M1 cells can be induced to differentiate into the monocyte/macrophage pathway by various inducers. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation inhibiting factors (I-factors) in a cell lysate and conditioned medium of differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have now purified one of the I-factors from conditioned medium of differentiation resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16000–17000 Da (16K I-factor). The amino acid sequence of all fragments of the 16K I-factor we have found are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor, a candidate suppressor protein for differentiation of leukemic cells, is Nm23/nucleoside diphosphate kinase protein.

When CEM cells were incubated with [3H]-labeled 3'-azido-2',3'-dideoxyguanosine (AzddGuo), the isotope was largely recovered as ribo- and deoxyribonucleotides in the acid soluble fraction of CEM cells, due to extensive catabolism of AzddGuo and recycling of the guanine formed by purine nucleoside phosphorylase. Only 10% was found as the AzddGuo nucleotides, with the diphosphate of AzddGuo as the dominating nucleotide at all time points. Thus nucleoside diphosphate kinase was rate limiting for the formation of AzddGuo triphosphate, responsible for the toxic and antiviral activity of the nucleoside. Inhibition of de novo deoxyribonucleotide synthesis with hydroxyurea increased the phosphorylation of AzddGuo twofold.

An in vitro study of phosphate-transfer, from the high-energy phosphates on the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinase to GDP on various guanine nucleotide binding proteins (G1, elongation factor alpha 1, recombinant v-rasH p21 protein, transducin, Gi and Go), revealed that the GDP acts as a phosphate-acceptor, in the presence of divalent cations (Mg2+ and Ca2+). This finding suggests that via phosphate-transfer, NDP-kinase may be responsible for the direct activation of various guanine nucleotide binding proteins through phosphate-transfer by the enzyme.

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 (i) 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 (Mg 2+ and Ca 2+ ) results in the formation of nucleoside 5′-triphosphates (NTPs) within 40 sec even at low temperatures (below 4°C) without strict base-specificity; and (ii) 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 Ca 2+ or 1 mM Mg 2+ 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.

Summary Ribonucleotide reductase from Escherichia coli contains nucleoside diphsophate kinase activity even in highly purified preparations. Although the bulk of the kinase activity was removed during the initial steps of the ribonucleotide reductase purifications some activity remained in the purest fractions. The nucleoside diphosphate kinase reaction is probably catalyzed by a protein different from ribonucleotide reductase. The tight binding between the two enzymes might reflect an association between them in the living cell.

In the presence of ATP, 1M-salt-washed ribosomes and subunits of both eukaryotes and prokaryotes phosphorylate GDP to 5′GTP. A possible role for the ribosome-associated GDP kinase is presented.

Effects of naturally occurring polyamines were tested on the activity of bovine liver nucleosidediphosphate kinase with ATP as phosphoryl donor and eight nucleosidediphosphates as phosphoryl acceptors. The enzyme was either stimulated, inhibited, or unaffected depending upon the nucleosidediphosphate substrate and the polyamine, indicating that a general cation effect is not a sole mechanism of polyamine action. This selectivity and specificity of the effects with respect to both the polyamines and the nucleosidediphosphates leads us to speculate that an action of polyamines on nucleosidediphosphate kinase may play a significant role in the regulation of specific nucleosidetriphosphate synthesis in vivo .

Summary The ADP analog α,β-methylene adenosine 5′-diphosphate was photophosphorylated to α,β-methylene adenosine 5′-triphosphate by spinach chloroplasts. The phosphorylation of the analog was not due to nucleotide diphosphokinase activity. The ADP analog was binding to the nucleotide binding sites on the photosynthetic membranes as well as to the solubilized coupling factor protein. Binding of ADP to the photosynthetic membranes was decreased in the presence of the ADP analog indicating a direct interaction of the ADP analog with the ADP binding sites on the thylakoids.

GTP-sensitive adenylate cyclases in liver membranes achieved by glucagon and by cholera toxin pretreatment displayed similar responses to added GTP in assay with respect to magnitude and sensitivity. However, their susceptibility to GTP formed during incubation from added GDP catalized by membrane-associated nucleoside diphosphate kinase(mNDPK) was different. Adenylate cyclase pretreated with cholera toxin was essentially unaffected by added GDP, while further addition of glucagon produced activation. GTP-stimulated adenylate cyclase activity in toxin-treated membranes was inhibited by added GDP, whereas glucagon addition reduced the inhibitory action of GDP by two orders of magnitude. Since neither pretreatment with toxin nor glucagon addition altered GTP formation by mNDPK, these observations suggest a possible presence of a mechanism by which hormone makes adenylate cyclase susceptible to the GTP formed via mNDPK for activation.

Guanosine and inosine diphosphate (GDP end IDP) have been shown to be possible phosphate acceptors in the succinic thiokinase reaction in mammalian kidney and heart, ( Mazumder, Sanadi and Rodwell 1960 ): Succinyl CoA + XDP -+ Succinat + XTP + CoA (1) X = inosine or guanosine The nucleotide specifity of reaction (1) has been studied only in isolated enzyme preparations. The difference of Km between guanine and inosine nucleotides was shown to be small. Thus the question as to which nucleotide system,(the inosine or the guanine system) is the physiological participant in the phosphate transfer reactions (1) remained open. In order to answer this question, the role of guanine and inosine nucleotides in intact liver mitochondria was studied by following the incorporation of P 32 into phosphate compounds of mitochondria. The basis of the experiments was the ultramicro scale ion exchange chromatography for separation of phosphate compounds ( Schnitger et al. 1959, Heldt,.1963 ). This was combined with simultaneous chart recording of ultraviolet absorption, analysis of total phosphorous content and radioactivity. Exact data on radioactivity were obtained by digital recording. The PT2 labelled compounds were identified by comparing the chromatographic behaviour of the collected fractions with added components on thin layer radio electrophoresis and

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly in vivo by modulating the GTP GDP ratio in the microtubule environment.

Activity of purified protocollagen proline hydroxylase was enhanced several fold by addition of nucleoside triphosphates (3 mM) to the assay medium, but nucleoside mono-and diphosphates were almost inactive. Pyrimidine nucleotides were less effective compared with purine nucleotides, among which GTP was the most effective. dATP and ATP analogues such as adenosine 5′-(β,γ-imino) triphosphate (AMP-PNP), adenosine 5′-(β,γ-methylene) triphosphate (AMP-PCP), etc. were inactive. ATP or GTP showed no additive effect on enzyme activity stimulated by dithiothreitol or bovine serum albumin.

2-beta-D-Ribofuranosylselenazole-4-carboxamide, a selenazole analog of the antitumor agent Tiazofurin, is severalfold more cytotoxic to murine tumor cells in culture than Tiazofurin. Like Tiazofurin, the cytotoxicity of the selenazole analog is reversed by exogenous guanosine, and both nucleosides specifically inhibit IMP dehydrogenase activity in cultured P388 cells. The dose-dependency for this inhibition correlates with the relative cytotoxicities of both drugs, indicating that a more potent inhibition of IMP dehydrogenase by the selenazole analog is primarily responsible for its increased cytotoxicity. The specific inhibition of the isolated enzyme by potential metabolites of the selenazole analog is discussed.

Commercial preparations of adenosine 5′-(β, γ-imino)triphosphate (App(NH)p) were found to be contaminated with a GTP-like substance(s) as well as a phosphate donor(s) for GDP. Thus, when these preparations were used as substrate with no purification, GDP was as effective as GTP in promoting PGE 1 stimulation of human platelet adenylate cyclase. With purified App(NH)p as substrate, the effect of PGE 1 with GDP was reduced but still observable, while that with GTP was unaltered. PGE 1 also caused a stimulation in the presence of guanosine 5′-o-(2-thiodiphosphate) (GDPβS) with ATP as substrate. Both of the PGE 1 -stimulated activities observed with GDP and its analog were completely lost by the addition of UDP, thereby, inhibiting GTP formation catalized by membrane-associated nucleoside diphosphate kinase. The results demonstrate that the stimulatory effects of PGE 1 observed with GDP and App(NH)p, and with GDPβS and ATP were transphosphorylation dependent and, therefore, the analogs must be used with special caution in adenylate cyclase studies.

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.