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PubMed1987

Structural and biochemical properties of bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate and adenosine 5'-diphosphate.

The structural and biochemical properties of the alpha,beta-bidentate tetraaquarhodium(III) complexes of inorganic pyrophosphate [Rh(H2O)4PP] and adenosine diphosphate [Rh(H2O)4ADP] are examined. These Rh(III) complexes are exchange-inert analogues of the corresponding physiologically important MgIIPP and MgIIADP complexes. The crystal structure of [Rh(H2O)4H2P2O7]+Cl- shows that the six-membered chelate ring adopts a twist-boat conformation with an unusually high puckering amplitude of 0.756 (3) A. The Rh coordination distances average 2.02 (1) A, while the bridge P-O bonds are virtually equal in length. All 10 protons of the complex participate in hydrogen bonding. There are two intramolecular hydrogen bonds between the phosphate oxygen atoms and the axially coordinated water molecules. The Rh(H2O)4PP complex was found to be a substrate for yeast inorganic pyrophosphatase, with Ki = 0.063 (7) mM and Vm = 500 (100) min-1. The two screw sense isomers of Rh(H2O)4ADP were prepared from (Rp)-[alpha-16O,18O]ADP and assigned configuration on the basis of the magnitude of their 31P NMR isotopic chemical shifts. The Rh(H2O)4ADP complex binds a number of kinases as tightly as MgADP. Arginine kinase and creatine kinase were shown to bind the delta Rh(H2O)4ADP isomer 7 and 45 times tighter, respectively, than the lambda isomer. The reactivity of Rh(H2O)4PP with pyrophosphatase is comparable to that of Cr(H2O)4PP, and the binding affinities of the Rh(H2O)4ADP screw sense isomers for kinases are also comparable to those observed for the corresponding Cr(H2O)4ADP screw sense isomers.

Adenosine Diphosphate↗

PubMed1981

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid.

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.

Adenosine Diphosphate↗

PubMed1976

Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative.

The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.

Affinity Labels↗

PubMed1995

Mechanism of inhibition of poly(ADP-ribose) glycohydrolase by adenosine diphosphate (hydroxymethyl)pyrrolidinediol.

Adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD), a nitrogen-in-the-ring analog of ADP-ribose, was recently shown to be a potent and specific inhibitor of poly(ADP-ribose) glycohydrolase. Analysis of the inhibition kinetics of the hydrolase by ADP-HPD using the method of Lineweaver and Burk yields a noncompetitive double-reciprocal plot. Both the intercept (1/V) versus [inhibitor] replot and the slope (Km/V) versus [inhibitor] replot are hyperbolic, indicating partial noncompetitive inhibition. Inhibitor dissociation constants Kii = 52 nM and Kis = 80 nM were determined for ADP-HPD by analysis of the intercept versus [inhibitor] and slope versus [inhibitor] replots. These results show that although ADP-HPD is extremely potent in inhibiting poly(ADP-ribose) glycohydrolase, its effectiveness is limited by its partial inhibition. ADP-HPD was significantly less potent as an inhibitor of the NAD glycohydrolase from Bungarus fasciatus venom. Analysis of the inhibition kinetics using the Lineweaver and Burk method indicated that ADP-HPD was a linear-competitive inhibitor of the NAD glycohydrolase with a Ki of 94 microM. The results indicate that at low concentration ADP-HPD will be a selective inhibitor of poly(ADP-ribose) glycohydrolase; however, complete inactivation of the activity will be difficult to obtain.

Adenosine Diphosphate↗

PubMed2003

SAR analysis of adenosine diphosphate (hydroxymethyl)pyrrolidinediol inhibition of poly(ADP-ribose) glycohydrolase.

Polyadenosine diphosphoribose glycohydrolase (PARG) catalyzes the intracellular hydrolysis of adenosine diphosphoribose polymers. Because structure-activity data are lacking for PARG, the specific inhibitor adenosine diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) was utilized to determine the effects of structure on inhibitor potency using PARG isolated from bovine thymus (bPARG) and recombinant bovine PARG catalytic fragment (rPARG-CF). Both enzymes were strongly inhibited by submicromolar levels of ADP-HPD, but ADP and the phosphorylated pyrrolidine displayed no activity. Utilizing ADP-HPD analogues containing 2-, N(6), or 8-adenosyl substituents or guanine instead of adenine, the importance of adenine ring recognition as well as a correlation between loss of PARG inhibition and the length and bulkiness of 8-adenosyl substituents was shown. Utilization of ADP-HPD analogues lacking one or both pyrrolidine cis-hydroxyls demonstrated their importance for inhibitor binding. Last, the similarity between naturally occurring bPARG and heterologously expressed rPARG-CF was demonstrated. Therefore, readily available rPARG-CF is suitable for use in future studies to determine the structural aspects of PARG.

Adenosine Diphosphate↗

PubMed2000

Regulation of cellular functions by nucleoside diphosphate kinases in mammals.

The role of nucleoside diphosphate (NDP) kinases in cell growth, differentiation, and tumor metastasis in relation to signal transduction was investigated. The essential role of NDP kinase in cell growth was validated by coupling between reduced NDP kinase levels, induced by antisense oligonucleotides, and the suppression of proliferative activity of a cultured cell line. In addition, because NDP kinase levels are often enhanced with development and differentiation, as has been demonstrated in postmitotic cells and tissues, such as the heart and brain, we further examined this possibility using the bone tissue (osteoblasts) and a cultured cell line PC12D. The enhanced NDP kinase accumulation was demonstrated in the matured osteoblasts in vivo and in vitro by immunohistochemistry. In PC12D cells, neurite outgrowth took place in NDP kinase beta-transfected clones without differentiation inducers, which was accompanied by prolongation of doubling time. Neurite outgrowth, triggered by nerve growth factor and a cyclic AMP analog, was down-regulated upon forced expression of inactive mutant NDP kinase by virtue of a dominant negative effect. NDP kinase alpha-transfected rat mammary adenocarcinoma cells (MTLn3) and nm23-H2-transfected human oral squamous cell carcinoma cells (LMF4) manifested reduced metastatic potential and were associated with an altered sensitivity to environmental factors, such as motility and growth factors. NDP kinase alpha, compared to NDP kinase beta, was involved in a wide variety of the cellular phenomena examined. Taken together, NDP kinase isoforms appear to elicit both their own respective and common effects. They may have an ability to lead cells to both proliferative and differentiated states by modulating responsiveness to environmental factors, but their fate seems to depend on their surrounding milieu.

Amino Acid Sequence↗

PubMed2003

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter.

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.

Blotting, Western↗

PubMed1976

Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation.

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

Adenosine Diphosphate Sugars↗

PubMed1994

Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with nucleoside diphosphate kinase.

The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.

Adenosine Diphosphate Ribose↗

PubMed1999

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.

Animals↗

PubMed2001

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.

Amino Acid Sequence↗

PubMed1978

Uridine diphosphate N-acetylglucosamine stimulates uptake of nutrients by quiescent BALB/C3T3 cells.

Preincubation of quiescent BALB/C3T3 cells with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) in conditioned medium resulted in a time- and concentration-dependent stimulation of uptake of 2-deoxyglucose, uridine, and alpha-aminoisobutyric acid over that seen in conditioned medium alone. The apparent Km values for 2-deoxyglucose and uridine uptake were the same for cells incubated with or without UDP-GlcNAc, whereas the Vmax values were higher for cells pretreated with the nucleotide sugar. The stimulation of uptake was specific for UDP-GlcNAc, and the other nucleotide sugars tested were ineffective; incubation of UDP-GlcNAc-pretreated cells with N-acetylglucosaminidase abolished the stimulatory effect. In all cases, the extent of stimulation of nutrient uptake was comparable to that seen with serum-stimulated cells. Incubation of quiescent cells in situ with UDP-[3H]GlcNAc led to incorporation of radioactive N-acetylglucosamine into the acid-precipitable fraction; a large fraction of the labeled amino sugar was found on the cell surface acceptors. We interpret these data to mean that the cellular acceptors of quiescent cells are "under-glycosylated," at least in terms of N-acetylglucosamine, and that stimulation of uptake of nutrients may be a consequence of restoration of the amino sugar residues on the oligosaccharide chains of acceptors on the cell surface.

Acetylglucosamine↗

PubMed1997

AlF3 mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate kinase and MgADP.

Nucleoside diphosphate kinase reversibly transfers the gamma-phosphate of ATP onto its active site histidine. We have investigated the transition state of histidine phosphorylation with the high-resolution crystal structures of the enzyme from Dictyostelium discoideum with MgADP and either aluminium or beryllium fluoride. The bound aluminium fluoride species is the neutral species AlF3 and not the more common AlF4-. AlF3 forms a trigonal bipyramid that makes it an accurate analog of the transition state of the gamma-phosphate of ATP undergoing transfer to the catalytic histidine. Its axial ligands are a histidine nitrogen and a beta-phosphate oxygen. Beryllium fluoride also binds at the same position and with the same ligands but in a tetrahedral geometry resembling the Michaelis complex rather than the transition state. The two x-ray structures show explicit enzyme-substrate interactions that discriminate between the ground and the transition states of the reaction. They also illustrate the partially dissociative geometry of the transition state of phosphoryl transfer and demonstrate the potential applications of metallofluorides for the study of kinase mechanisms.

Adenosine Diphosphate↗

PubMed1996

Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction.

The eukaryotic nucleoside diphosphate (NDP) kinases are hexamers, while the bacterial NDP kinases are tetramers made of small, single domain subunits. These enzymes represent an ideal model for studying the effect of subunit interaction on protein stability. The thermostability of NDP kinases of each class was studied by differential scanning calorimetry and biochemical methods. The hexameric NDP kinase from Dictyostelium discoideum displays one single, irreversible differential scanning calorimetry peak (Tm 62 degrees C) over a broad protein concentration, indicating a single step denaturation. The thermal stability of the protein was increased by ADP. The P105G substitution, which affects a loop implicated in subunit contacts, yields a protein that reversibly dissociates to folded monomers at 38 degrees C before the irreversible denaturation occurs (Tm 47 degrees C). ADP delays the dissociation, but does not change the Tm. These data indicate a "coupling" of the quaternary structure with the tertiary structure in the wild-type, but not in the mutated protein. We describe the x-ray structure of the P105G mutant at 2.2-A resolution. It is very similar to that of the wild-type protein. Therefore, a minimal change in the structure leads to a dramatic change of protein thermostability. The NDP kinase from Escherichia coli behaves like the P105G mutant of the Dictyostelium NDP kinase. The detailed study of their thermostability is important, since biological effects of thermolabile NDP kinases have been described in several organisms.

Adenosine Diphosphate↗

PubMed1997

A 16-kDa protein functions as a new regulatory protein for Hsc70 molecular chaperone and is identified as a member of the Nm23/nucleoside diphosphate kinase family.

Cytoplasmic Hsc70 is a multifunctional molecular chaperone. It is hypothesized that accessory proteins are used to specify the diverse chaperone activities of Hsc70. A 16-kDa cytosolic protein (p16) co-purified with Hsc70 obtained from a fish hepatocyte cell line, PLHC-1. Hsc70 also co-immunoprecipitated with p16 from PLHC-1 cells and fish liver. p16 was identified as a member of the Nm23/nucleoside diphosphate (NDP) kinase family based on its amino acid sequence similarity, NDP kinase activity, and recognition by anti-human NDP kinase-A antibody. This antibody also co-immunoprecipitated Hsc70 and NDP kinase from human HepG2 cells. p16 monomerized Hsc70 and released Hsc70 from pigeon cytochrome c peptide (Pc) but not from FYQLALT, a peptide specifically designed for high affinity binding. Therefore, p16 may modulate Hsc70 function by maintaining Hsc70 in a monomeric state and by dissociating unfolded proteins from Hsc70 either through protein-protein interactions or by supplying ATP indirectly through phosphate transfer. p16 did not affect basal or unfolded protein-stimulated ATPase activity of bovine brain Hsc70 using in vitro assays. Interestingly, bovine liver NDP kinase did not dissociate the Hsc70.Pc complex. In addition, two nonconservative amino acid subsitutions were found near the amino terminus of p16. Therefore, p16 may be a unique Nm23/NDP kinase that functions as an accessory protein for cytosolic Hsc70 in eukaryotes.

Adenosine Triphosphatases↗

PubMed1998

Decrease in nucleoside diphosphate kinase (NDPK/nm23) expression during hematopoietic maturation.

The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34(+) progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells.

Cell Differentiation↗

PubMed1998

Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. Two old enzymes combine for the novel Nm23 protein phosphotransferase function.

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

Creatine Kinase↗

PubMed1999

Cleavage of DNA by human NM23-H2/nucleoside diphosphate kinase involves formation of a covalent protein-DNA complex.

The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.

Antigens, Neoplasm↗