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PubMed1995

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors.

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.

Abciximab↗

PubMed1988

Early changes in the substructure of the marginal bundle in human blood platelets responding to adenosine diphosphate.

Human blood platelets were exposed to 10 microM adenosine diphosphate in order to induce shape changes and their ultrastructure was examined in preparations fixed with a glutaraldehyde-tannic acid mixture. Within 5 sec after the addition of ADP the marginal bundle along the perimeter of the platelet had undergone profound changes. The intertubular filaments of the marginal bundle were lost and, probably as a consequence, the microtubules were dispersed. The microtubules also had fragmented and appeared as straight rods rather than smoothly curved coils. Profiles with 14 or 15 protofilaments rather than the common 13-unit pattern were seen in 16% of the cross-sectioned microtubules. The proportion of incomplete (C-shaped) microtubules or of microtubules with an electron-dense interior also had increased and were interpreted as sections near the terminal ends of a microtubule. We interpret the C-shaped and the "filled" microtubules as signs of a disassembled microtubule and the 14- and 15-protofilament profiles as probably representing new (ends of) microtubules reassembled with a defective control of the proper structure.

Adenosine Diphosphate↗

PubMed1994

Dualistic behavior of ATP-sensitive K+ channels toward intracellular nucleoside diphosphates.

ATP-sensitive K+ (KATP) channels are intracellular ligand-gated channels which regulate diverse cellular functions. Intracellular nucleoside diphosphates (NDPs) are essential for the physiological opening of KATP channels which would otherwise be permanently closed by their overt sensitivity to intracellular ATP. We find that KATP channels exhibit dualistic behavior toward NDPs depending on their operative condition. When channels are in the spontaneous operative condition, NDPs antagonize channel inhibition by intracellular ATP. When channels have "run down", NDPs induce channel opening but no longer antagonize intracellular ATP. The switch of the KATP channel response to the same ligand, i.e., NDPs, is controlled by a Mg-ATP-dependent reaction. The condition of the target protein therefore determines the effect of the ligand. This property provides a novel basis to evaluate the dynamic regulation of ion channels by their ligands.

Adenosine Triphosphate↗

PubMed1992

Effects of orally administered cytidine 5'-diphosphate choline on brain phospholipid content.

Cytidine, as cytidine 5'-diphosphate choline (CDP-choline), is important for the synthesis of phosphatidylcholine in cell membranes. To investigate whether exogenous CDP-choline could affect brain phospholipid composition, we supplemented the diet of mice with this drug (500 mg/kg/day) for 27 months in 3-month-old mice and for 90, 42, and 3 days in 12-month-old mice, and measured their levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and the content of phosphatidylinositol plus phosphatidic acid in the cerebral cortex. After 27 months of treatment, PC and PE increased significantly by 19% (P < 0.05) and by 20% (P < 0.01), respectively. PS levels increased by 18% (not statistically significant). Similar elevations in PC and PE levels were obtained when older mice were treated for only 3 months (P < 0.05). No changes were observed with shorter treatment periods. These results suggest that chronic administration of CDP-choline can have effects on brain phospholipid composition that may underlie its reported utility in various neurologic disorders.

Administration, Oral↗

PubMed1993

Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila.

BACKGROUND: Nucleotide diphosphate kinase (NDP kinase) is a phosphate transfer enzyme involved in cell regulation and in animal development. Drosophila NDP kinase is the product of the abnormal wing disc (awd) developmental gene, a point mutation in which can produce the killer of prune (K-pn) conditional lethal phenotype. The highly homologous mammalian genes control metastasis and a human NDP kinase acts as a transcription factor. RESULTS: The X-ray structure of the Awd protein prepared from Drosophila was solved at 2.4 A resolution by molecular replacement from the homologous Dictyostelium protein. Both are hexamers, and both have the same fold and the same active site. Subunit contacts differ as a result of sequence changes in the carboxy-terminal segment and in the loop that is the site of the K-pn mutation. CONCLUSIONS: Regulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.

Amino Acid Sequence↗

PubMed1996

Phytochrome regulates phosphorylation of a protein with characteristics of a nucleoside diphosphate kinase in the crude membrane fraction from stem sections of etiolated pea seedlings.

The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [gamma-32P]ATP at 4 x 10(-8) M for 15 s at 0 degree C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 mumol m-2 s-1) for 20 s. Successive irradiation with red light (100 mumol m-2 s-1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 mumol m-2 s-1 for 180 s). A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 x 10(-8) M [gamma-32P]ATP at 0 degree C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10(-5) or 10(-6) M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and crossreactivity with an antibody specific to ricet NDP kinase as assayed by immunoprecipitation.

Animals↗

PubMed2004

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase.

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.

Adenosine Diphosphate Glucose↗

PubMed2005

Extraction and identification by mass spectrometry of undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc from Escherichia coli.

Undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc (lipid II) is extracted from Escherichia coli cells by utilizing its unusual pH-dependent solubility property in a Bligh-Dyer system, and identified by electrospray ionization mass spectrometry in conjunction with a novel 15N mass shift analysis. The described approach will facilitate the structural characterization of lipid II variants from diverse bacteria, including antibiotic-resistant mutants, as well as the numerous minor uncharacterized lipids present in all biological systems.

Diphosphates↗

PubMed2006

In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner.

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC 2.7.4.3) and nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-gamma-lactone dehydrogenase (GLDH) (EC 1.3.2.3), the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.

Adenylate Kinase↗

PubMed2004

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography.

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.

Adenosine Diphosphate↗

PubMed2004

Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA.

E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.

Base Sequence↗

PubMed2004

Facilitated folding and subunit assembly in Escherichia coli and in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag.

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 degrees C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.

Archaea↗

PubMed2005

Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum.

Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate-bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2A, respectively. Crystals with the apo-form were obtained with His6-tagged enzyme, whereas the untagged form was used for co-crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6-tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.

Crystallization↗

PubMed2006

Interaction of nucleoside diphosphate kinase and catalases for stress and light responses in Neurospora crassa.

Nucleoside diphosphate kinase (NDK) is an ubiquitous enzyme with the function of a signal transducer. In Neurospora crassa, an ndk-1(P72H) mutant carrying the point mutation Pro72His was isolated. We found that ndk-1(P72H) showed hypersensitivity to oxidative and heat stress and a decrease in the levels of catalase (Cat)-1 and -3 induced by oxidative, heat stress and illumination compared with wild type (Wt). We found, by conducting a yeast two-hybrid assay, that Cat-1 interacted with NDK-1. NDK-1 was suggested to control Cat-1 and Cat-3 at the post-transcriptional level in response to heat, oxidative stress and light.

Catalase↗

PubMedDate not supplied

Evaluation of adenosine 5'-diphosphate (ADP)- and collagen-induced platelet aggregation in canine leishmaniasis.

Leishmania-infected dogs, which represent an important reservoir of infection in many parts of the world, frequently suffer from haematological disorders, including thrombocytopenia. In this study, the ability of platelets from healthy (control) dogs (n = 11) and from dogs with naturally acquired clinical leishmaniasis (n = 24) to aggregate in the presence of two different agonists (adenosine 5'-diphosphate [ADP] and collagen) was assayed. Haematological parameters examined consisted of the platelet count, prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen concentration and D-dimer concentration. In dogs with leishmaniasis, a significant decrease in ADP- and collagen-induced platelet aggregation was observed. Compared with platelets from the control dogs, those from leishmania-infected dogs showed a higher sensitivity to collagen, as demonstrated by a reduction in platelet aggregation of up to 20.4%, and a significant (P < 0.0001) difference for all the doses tested. With ADP the reduction was up to 10.4%, the difference reaching a significant level of P < 0.0001 only at the maximum dose used. The nature of this response, which was not accompanied by any clinical signs of bleeding other than an increase in aPTT, emphasizes the role of platelets in the parasite-host cell interaction.

Adenosine Diphosphate↗

PubMed2004

Coordination mode of adenosine 5'-diphosphate in ternary systems containing Cu(II), Cd(II) or Hg(II) ions and polyamines.

The interactions of adenosine 5'-diphosphate (ADP) with some polyamines (PA) (1,3-diaminopropane (tn), 1,4-diaminobutane (Put), 1,7-diamino-4-azaheptane (3,3-tri) and 1,8-diamino-4-azaoctane (Spd)) both in presence and in the absence of metal ions (Cu(II), Cd(II) and Hg(II)) have been studied. In the metal-free systems the formation of adducts (ADP)Hx(PA) has been observed, in which the main reaction centres are the endocyclic nitrogen atoms of the purine ring, the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine. The effectiveness of the phosphate group in formation of adducts has been found to decrease in the series Put > Spd > Spm and to be lower than in the reactions with shorter homologues of biogenic amines. In the ternary systems with metal ions the formation of molecular complexes (ML L' type) has been evidenced in which the protonated polyamine interacts with the nitrogen atoms N(1) or N(7) of the purine ring of the nucleotide. In the ternary systems Cu(II)/ADP/polyamine the coordination dichotomy observed in the binary system Cu(II)/ADP disappears. In the systems with Hg(II) ions the pH range of the dichotomy is extended, while for the systems Cd(II)/ADP/polyamine no changes of the range relative to the binary system Cd(II)/ADP have been noted.

Adenosine Diphosphate↗

PubMed2005

Interaction centres of pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) in their reactions with tetramines and Cu(II) ions.

The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and 1,11-diamino-4,8-diazaundecane (3,3,3-tet) have been studied. The composition and stability constants of the complexes formed have been determined by means of the potentiometric method, while the centres of interactions in the ligands have been identified by the spectral methods (UV-Vis, Ultraviolet and Visible spectroscopy; EPR, electron spin resonance; NMR). In the systems without metal, formation of the molecular complexes nucleotide-polyamine with the interaction centres at the endocyclic nitrogen atom of purine ring N3, the oxygen atoms of the phosphate group from the nucleotide and protonated nitrogen atoms of the polyamine have been detected. Significant differences have been found in the metallation between the systems with Spm and with 3,3,3-tet. In the systems with spermine, mainly protonated species are formed with the phosphate group of the nucleotide and deprotonated nitrogen atoms of the polyamine making the coordination centres, while the donor nitrogen atom of the nucleotide N3 is involved in the intramolecular interligand interactions, additionally stabilising the complex. In the systems with 3,3,3-tet, the MLL' type species are formed in which the oxygen atoms of the phosphate group and nitrogen atoms of the polyamine are involved in metallation, whereas the N3 atom from the pyrimidine ring of the nucleotide is located outside the inner coordination sphere of copper ion. The main centre of Cu(II) interaction in the nucleotide, both in the system with Spm and 3,3,3-tet is the phosphate group of the nucleotide.

Amines↗

PubMed2004

Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase-2 for phytochrome-mediated light signaling.

In plants, nucleoside diphosphate kinases (NDPKs) play a key role in the signaling of both stress and light. However, little is known about the structural elements involved in their function. Of the three NDPKs (NDPK1-NDPK3) expressed in Arabidopsis thaliana, NDPK2 is involved in phytochrome-mediated signal transduction. In this study, we found that the binding of dNDP or NTP to NDPK2 strengthens the interaction significantly between activated phytochrome and NDPK2. To better understand the structural basis of the phytochrome-NDPK2 interaction, we determined the X-ray structures of NDPK1, NDPK2, and dGTP-bound NDPK2 from A.thaliana at 1.8A, 2.6A, and 2.4A, respectively. The structures showed that nucleotide binding caused a slight conformational change in NDPK2 that was confined to helices alphaA and alpha2. This suggests that the presence of nucleotide in the active site and/or the evoked conformational change contributes to the recognition of NDPK2 by activated phytochrome. In vitro binding assays showed that only NDPK2 interacted specifically with the phytochrome and the C-terminal regulatory domain of phytochrome is involved in the interaction. A domain swap experiment between NDPK1 and NDPK2 showed that the variable C-terminal region of NDPK2 is important for the activation by phytochrome. The structure of Arabidopsis NDPK1 and NDPK2 showed that the isoforms share common electrostatic surfaces at the nucleotide-binding site, but the variable C-terminal regions have distinct electrostatic charge distributions. These findings suggest that the binding of nucleotide to NDPK2 plays a regulatory role in phytochrome signaling and that the C-terminal extension of NDPK2 provides a potential binding surface for the specific interaction with phytochromes.

Amino Acid Sequence↗