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PubMed1994

Recombinant rat nucleoside diphosphate kinase isoforms (alpha and beta): purification, properties and application to immunological detection of native isoforms in rat tissues.

We previously demonstrated that at least two isoforms of nucleoside diphosphate (NDP) kinase, the products of two different tandemly arrayed genes, are present in rat. To understand the physiological role of each isoform, some biochemical properties of recombinant rat NDP kinase alpha- and beta-isoforms, produced in large amount, were studied. cDNAs of the two isoforms were inserted in an expression vector pET3b and recombinant enzymes were overproduced in Escherichia coli. Their primary structures were different from the native enzymes in that the latter suffer from modification of the NH2-terminal end. The two recombinant isoforms were purified from the cell lysate to apparent homogeneity by ammonium sulfate fractionation followed by three successive column chromatographies. Despite their extreme similarity in the amino-acid sequences, the two showed somewhat different enzymic properties in terms of di- and triphosphate nucleotide substrate specificity. They showed similar mobilities on SDS-PAGE as expected from their calculated molecular weight (alpha-isoform, 17,283 versus beta-isoform, 17,192) but differed in isoelectric point (alpha-isoform, pI 6.7; beta-isoform, pI 7.8) and heat stability. Polyclonal antibody which reacted with both isoforms and alpha-isoform-specific monoclonal antibodies differentially recognized native enzymes from rat tissues after the tissue extracts were separated by isoelectric focusing gel electrophoresis under a denaturation condition. The results showed that the alpha-isoform, though its amount varied from one tissue to another, was the major form in rat tissues examined compared with the beta-isoform which was detectable in brain and testis. There was no preference in their subcellular localization when examined with myelin, synaptosomal supernatant and total homogenate fractions from the rat cerebrum and cerebellum.

Amino Acid Sequence↗

PubMed1996

Rapid purification of a chloroplast nucleoside diphosphate kinase using CoA-affinity chromatography.

An 18 kDa protein from spinach chloroplasts was purified in one step to homogeneity using CoA-affinity chromatography. Its N-terminal sequence was identical to spinach nucleoside diphosphate kinase II (NDPK II). The kinase was isolated as a approximately 100 kDa complex. Immunoblotting detected NDPKII in plastids from leaves, roots, seeds and male flowers. NDPK I, an isoform of NDPK II, was not found in chloroplast soluble extracts, whereas NDPK III was present.

Amino Acid Sequence↗

PubMed1988

Guanosine diphosphate binding, metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes.

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [3H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a Ka of 4.5.10(6) M-1 and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5'-O-[3-thio]triphosphate (Gppp[S]), was most effective as an inhibitor of [3H]GDP binding (ED50 = 4.8.10(-8) M), whereas guanosine 5'-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED50 = 3.4.10(-7) M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg2+ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P less than 0.05) Gppp[S]-stimulated adenylate cyclase activity (Ki = 1.8.10(-7) M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a Koff value of 6 +/- 1.min-1. Using a two-stage pre-incubation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.

Adenylyl Cyclases↗

PubMed1995

Effect of retinoic acid on Nm/23 nucleoside diphosphate kinase and components of cyclic adenosine monophosphate-dependent signalling in human neuroblastoma cell lines.

The effects of retinoic acid on components of the cAMP-dependent signalling system were examined in two related human neuroblastoma cell lines SK-N-SH-F (SHF) and SK-N-SH-N (SHN). Retinoid treatment for a week significantly increased the concentration of intracellular cAMP and the levels of activity of protein kinase A and adenylate cyclase in both cell lines. Retinoic acid treatment also caused a very marked translocation of nucleoside diphosphate kinase from the cytosol to the membrane fraction. The increases in cyclic nucleotide and protein kinase A activity were observed to occur as early as within 1 and 2 days respectively and preceded or were concurrent with the onset of observable morphological differentiation. Results also indicated that agents which elevated intracellular cAMP caused neuronal differentiation and blunted retinoic acid-induced melanocytic differentiation in SHF cells. However, increases in cAMP brought about by treatment of SHF cells with retinoic acid alone were several-fold smaller and thus insufficient to induce neuritogenesis in these cells. The results as a whole indicate that one overall effect of retinoic acid treatment is to upgrade the activity of components of the cAMP-dependent signalling system in both neuroblastoma cell lines. However, retinoic acid causes the SH-F and SH-N cell lines to differentiate along different routes which means that the upgrading responses may be related to more general aspects of differentiation rather than to specific phenotype expression.

Adenylyl Cyclases↗

PubMed1996

Neutralizing antibodies to nucleoside diphosphate kinase inhibit the enzyme in vitro and in vivo: evidence for two distinct mechanisms of activation of atrial currents by ATPgammaS.

Nucleoside diphosphate kinase (NDPK) participates in multiple cellular functions, yet the molecular mechanisms of its involvement are often unknown, given that there are no specific inhibitors for the enzyme from vertebrates. We developed antibodies against NDPK by immunization of rabbits with the enzyme from bullfrog skeletal muscle. The antibodies specifically recognized the enzyme from frog tissues, and cross-reacted with NDPK from Xenopus. In contrast to mammalian NDPK, the amphibian enzyme elicited antibodies that inhibit potently its catalytic function. We utilized the inhibitory properties of these immunoglobulins to examine the role of NDPK on the ATPgammaS-induced stimulation of Ca2+ and K+ currents of cardiac myocytes. Injection of NDPK-neutralizing Fab fragments into atrial cells reduced considerably the effect of ATPgammaS on muscarinic K+ currents, but not on Ca2+ currents. Therefore, ATPgammaS increases calcium and potassium currents of atrial cells by two distinct mechanisms. NDPK is essential for the conversion of ATPgammaS into GTPgammaS which leads to muscarinic K+ channel activation but not for the stimulation of Ca2+ currents by ATPgammaS. The results demonstrate that antibodies to frog NDPK block the activity of the enzyme in vivo and in vitro, and can be used to determine the relevance of NDPK and its catalytic activity to the function of vertebrate cells.

Adenosine Triphosphate↗

PubMed1991

The effect of calcitonin gene-related peptide on arrhythmia caused by adenosine diphosphate and desacetyldigilanide-C in rats.

We investigated the effect of calcitonin gene-related peptide on the arrhythmia induced by two drugs in rats. The results of our experiments have proved that calcitonin gene-related peptide could reduce the degree of atrioventricular block, protect against the attacks of sinus standstill and ventricular fibrillation produced by adenosine diphosphate, and improve restoration of sinus rhythm. Calcitonin gene-related peptide was able to eliminate sinus standstill and ventricular fibrillation resulting from administration of desacetyldigilanide-C. These results demonstrate that calcitonin gene-related peptide has strong antiarrhythmic effects in experimental animals.

Adenosine Diphosphate↗

PubMed1991

Overexpression of nucleoside diphosphate kinase (Nm23) in solid tumours.

The product of the nm23-H1 gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P less than 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.

Blotting, Western↗

PubMedDate not supplied

Enzymatic polymerization of 5-mercuriuridine-5'-diphosphate with polynucleotide phosphorylase from E. coli.

We report the polymerization of 5-mercuriuridine-5'-diphosphate (ppUHgX), in the presence of an excess of beta-mercaptoethanol, with polynucleotide phosphorylase from E. coli. A degradation of mercurated nucleotides with mercaptans was observed and about 30% of incorporation of mercury in the polymer was obtained after treatment with pancreatic RNase. The influence of ppUHgX and pUHgX with or without beta-mercaptoethanol was also studied on the polymerization of UDP. The ppUHgX did not polymerize in the absence of beta-mercaptoethanol.

Chromatography, Thin Layer↗

PubMed1996

Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation.

Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi state while the latter resembles more the S1.ATP state. In this work, the conformations of the S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx, complexes were examined by nucleotide chase and collisional quenching experiments. epsilon ADP release from S1.epsilon ADP.AlF4- was slower than that from S1.epsilon ADP.BeFx. However, acrylamide titrations of S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx showed little difference in nucleotide protection from quenching between the two complexes. This contrasts with the earlier observation on phosphate analog-dependent changes in the reactivity of the SH1 group on S1. To confirm phosphate-related perturbation of the SH1-SH2 sequence, emission spectra of fluorescein (IAF)-labeled SH1 and IANBD-labeled SH2 were recorded for S1 complexes with nucleotides and phosphate analogs. Considerable differences were found between the BeFx and AlF4- complexes with S1.MgADP for both SH1- and SH2-labeled proteins. These results are consistent with a recent crystallographic study of S1 complexes with ADP and phosphate analogs [Fisher et al., Biophys. J., 68 (1995) 19S] and the idea that the opening of the nucleotide cleft on S1 does not change much during ATP hydrolysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12720], while significant changes in the SH1-SH2 region accompany phosphate cleavage.

Acrylamides↗

PubMed1977

Plant glycoprotein biosynthesis. Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root.

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radio-labeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-D-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,

Glycoproteins↗

PubMed1988

The effect of fructose 1,6 diphosphate on pyruvate kinase from the liver of the flounder (Platichthys flesus L.).

1. Pyruvate kinase purified from flounder liver in two forms, i.e. PKI and PKII, is activated by fructose 1,6 diphosphate. 2. Two or more binding sites for FDP are demonstrated for PKII, the binding to which is influenced by the levels of substrates. 3. FDP reduces or abolishes the cooperative effect of PEP. 4. FDP increases the maximal activity. 5. The inhibition observed at higher levels of ADP is not abolished by FDP.

Adenosine Diphosphate↗

PubMed1995

The compartmentation of nucleoside diphosphate kinase in mitochondria.

The compartmentation of nucleoside diphosphate kinase (NDPK) was studied in mitochondria isolated from heart and liver of rat, rabbit, and pigeon. Compartmentation was assessed by determining latencies of enzyme activities, fractionating mitochondria with digitonin, and treating mitochondria with trypsin in the presence and absence of digitonin. NDPK activity in pigeon liver mitochondria was five- and seven-fold higher than in rat and rabbit liver mitochondria. The ratios of NDPK activities in liver vs. heart mitochondria were about 15 for rat, 2 for rabbit, and more than 40 for pigeon. Nearly all NDPK in pigeon liver mitochondria is in the matrix space, but outside the matrix in rat and rabbit liver mitochondria. Most NDPK in pigeon heart mitochondria was located outside the matrix while a significant fraction may be in the matrix of rat and rabbit heart mitochondria. These results are discussed relative to the assumed role that mitochondrial NDPK transfers the phosphoryl group of GTP produced in the Krebs cycle to the adenine nucleotide pool.

Animals↗

PubMed1991

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.

Amino Acid Sequence↗

PubMed1993

Isolation, overexpression and disruption of a Saccharomyces cerevisiae YNK gene encoding nucleoside diphosphate kinase.

Nucleoside diphosphate kinase (NDPK) is the major enzyme responsible for the synthesis of all nucleoside triphosphates except ATP. A gene (YNK) encoding NDPK was isolated from the yeast Saccharomyces cerevisiae. The coding region consists of 459 bp encoding 153 amino acid (aa) residues. The M(r) of NDPK, calculated from the deduced aa sequence, is 17,166. Yeast NDPK was 59% and 58% identical to the rat NDPK alpha and beta, respectively. Overexpression of YNK in yeast showed high NDPK activity. The overproduced NDPK cross-reacted with anti-NDPK antibody raised against rat NDPK by Western blot analysis. Despite the fact that NDPK has features of a housekeeping enzyme, disruption of the YNK locus in a haploid strain was neither lethal nor significantly affected phenotypic behaviors such as growth rate, spore formation, mating ability and morphology. Yeast with a defective YNK still possessed NDPK activity to approximately 10% of the wild-type level. Possible sources of the remaining enzyme activity are discussed.

Amino Acid Sequence↗

PubMed1994

Bacteriophage T4 ribonucleoside diphosphate reductase: on the defect causing decreased formation of the beta 93(2) subunit encoded by the nrdB93 mutant gene.

Bacteriophage T4 ribonucleoside diphosphate reductase is composed of two proteins, alpha 2 and beta 2, encoded by the nrdA and nrdB genes, respectively. The expression of nrdB is the limiting factor for the assembly of the enzyme. A recently described mutation, nrdB93, may give new insight into the regulation of synthesis of the beta subunit encoded by nrdB. Infection by T4 nrdB93 produced only low concentrations of the beta 2(93) protein. However, a site-specific mutation of phage T4 gene 39, encoding one of the subunits of T4 DNA topoisomerase, phenotypically suppressed the defect. The present work sought to characterize the nature of this defect. The mutation in nrdB93 was a single-base transition (G-->A) resulting in a Gly253-->Asp change. In vivo and in vitro studies provided no evidence of degradation of the beta 2(93) protein. Furthermore, the decrease in beta 2(93) formation was not caused by a delayed onset of transcription, neither by a decreased rate of mRNA formation from the nrdB promoter, nor by a defective intron splicing of the nrdB gene or in the transcription of the terminal segments of the message. These findings are consistent with the concept that the nrdB93 lesion produces a defect at the level of translation.

Amino Acid Sequence↗

PubMed1995

Nucleoside diphosphate kinase from the parasitic nematode Brugia malayi.

Using a reverse transcription-polymerase chain reaction (RT-PCR) procedure that exploited the presence of a conserved 22-nucleotide spliced leader (SL) sequence that is trans-spliced to the 5' end of nematode transcripts, a novel Brugia malayi (Bm) infective-stage SL cDNA expression library was constructed and characterized. The library was immunoscreened with rabbit anti-infective-stage antibodies (Ab) and an immunodominant clone, BmG4-7, was identified and characterized. BmG4-7 contained a full-length cDNA that had significant sequence similarity to nucleoside diphosphate kinase (NDK)-encoding sequences reported from a number of species, including Drosophila melanogaster and humans. BmNDK was found to be constitutively transcribed during all stages of parasite development. An anti-BmNDK Ab was used to immunostain a Western blot of extracts from adult and larval parasites. The Ab specifically recognized a 17.5-kDa molecule in all of the parasite extracts. Molecular modeling of the BmNDK showed several regions surrounding the conserved catalytic site that may be important in the design of drugs specific for the disruption of NTP synthesis in filarial parasites.

Amino Acid Sequence↗

PubMed1994

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Animals↗

PubMed1994

Capillary zone electrophoresis assay of the uridine diphosphate N-acetylmuramyl peptide precursors and the disaccharide pentapeptide derivative of bacterial cell wall peptidoglycan.

Uridine diphosphate N-acetylmuramyl peptide (EDP-MurNac) precursors and disaccharide pentapeptide of bacterial cell wall peptidoglycan were extracted from Enterobacter colacae cells and examined by capillary zone electrophoresis. Five UDP-MurNac derivatives with dibromopropamidine isethionate as the internal standard, and disaccharide pentapeptide with pyrimethamine as the internal standard, were successfully and rapidly analysed by using a fused-silica capillary and sodium phosphate buffer in methanol as the organic modifier at appropriate pH. Accurate quantitation was also achieved. The method provides the potential to investigate quantitatively the effect of antibacterials on the biosynthesis of peptidoglycan and to determine the relative cellular concentrations of the murein precursors within the cell cycle.

Cell Cycle↗