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PubMed1996

Quaternary structure of human nucleoside diphosphate kinase isoforms HA and HB in solution.

Human isoforms of nucleoside diphosphate kinase, NDPK-HA and NDPK-HB, have been expressed in E. coli and purified. Their apparent molecular masses have been determined by FPLC gel filtration. Absolute molecular masses were measured by equilibrium ultracentrifugation and sedimentation coefficients determined from the sedimentation velocity. Under near-physiological conditions, NDPK-HA has a mass of 101 +/- 3 kDa, close to that calculated for a hexamer (102.11 kDa), whilst NDPK-HB has a mass of 71 +/- 3 kDa, close to a tetramer (68.67 kDa). The sedimentation coefficients, 5.15 +/- 0.2 and 3.41 +/- 0.1 x 10(-13) s, for HA and HB also indicate a hexamer and a tetramer respectively. This suggests, although the crystal structure shows a hexameric quaternary arrangement [Webb et al. (1995) J. Mol. Biol. 251, 574-587], that NDPK-HB forms tetramers in solution like bacterial NDPK [Williams et al. (1993) J. Mol. Biol. 234, 1230-1247].

Chromatography, Gel↗

PubMed1986

Regulation of bilirubin glucuronide synthesis in primate (Macaca fascicularis) liver. Kinetic analysis of microsomal bilirubin uridine diphosphate glucuronyltransferase.

Hepatic bilirubin uridine diphosphate glucuronyl-transferase (UDP-glucuronyltransferase) catalyzes the formation of bilirubin monoglucuronides (BMG, C-8 and C-12 isomers) and bilirubin diglucuronide (BDG) from bilirubin and the cosubstrate, UDP-glucuronic acid. Distinctive patterns of bile pigment excretion occur in different species and in pathologic disorders (e.g., Gilbert's syndrome). In normal human and monkey (Macaca fascicularis) bile, the proportion of BDG exceeds that of BMG and the C-8/C-12 BMG isomer ratio approaches unity. To investigate the mechanisms responsible for the patterns of BDG and BMG isomers in bile, we used a radiochemical assay to analyze the kinetics and regulation of bilirubin UDP-glucuronyltransferase in microsomes prepared from monkey liver. The synthesis of BMG from bilirubin was a higher capacity, lower affinity step (Vmax = 295 pmol/mg protein X min, Km = 24 microM) than BDG synthesis from endogenously formed BMG (Vmax = 170 pmol/mg protein X min, Km = 14 microM). This observation was confirmed when biosynthetically prepared BMG was used as substrate. The rate of formation of BDG relative to BMG was modulated by both bilirubin and UDP-glucuronic acid concentration, whereas the C-8/C-12 BMG isomer ratio was influenced exclusively by UDP-glucuronic acid concentration. The data obtained with increasing UDP-glucuronic acid concentration did not obey conventional single-site kinetics, suggesting the presence of more than one binding site on the enzyme or a membrane transporter for this nucleotide sugar. These findings demonstrate that microsomal bilirubin UDP-glucuronyltransferase has a greater capacity for BMG than BDG synthesis, and thus support the concept that the decreased BDG/BMG ratio in the bile of patients with reduced hepatic enzyme activity (i.e., Gilbert's syndrome and type II Crigler-Najjar disease) reflects the diminished capacity of the enzyme to synthesize BDG from BMG. Bilirubin and UDP-glucuronic acid concentrations both appear to be pivotal in regulating the synthesis of individual bilirubin glucuronides and hence the patterns of bilirubin conjugates excreted in bile.

Animals↗

PubMed1991

Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum.

Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.

Crystallography↗

PubMed1989

Oxygen consumption and guanosine diphosphate binding by fetal brown adipose tissue in diabetic pregnancy.

Oxygen consumption and 3H-guanosine diphosphate (GDP) binding were determined in brown adipocytes and mitochondria from 28-day gestation fetuses of alloxan-diabetic rabbit does and saline-injected controls. Maternal diabetes was classified as severe or mild determined by whether maternal blood glucose values were greater or less than 200 mg/dL, respectively, at death. Basal oxygen consumption and adipocyte diameters did not vary among groups. A significant reduction in maximal norepinephrine (NE) stimulated O2 consumption by fetal brown adipose tissue (BAT) cells was seen in offspring of severely diabetic pregnancies when compared with control values (248 +/- 53 +/- v482 +/- 32 microL O2/10(6) cells/h; P less than .005). In contrast, a significant increase in maximal NE-stimulated O2 consumption by fetal BAT cells occurred in offspring of mild diabetic pregnancies (807 +/- 60, P less than .001 v controls). A highly significant inverse correlation between serum glucose levels and maximal O2 consumption by fetal BAT was observed in fetuses from mild and severe diabetic pregnancies (r = -.98, P less than .005), and there was no correlation between these two parameters in offspring of normal pregnancies. A significant inverse correlation was observed between maximal O2 consumption by fetal BAT cells and serum insulin levels in offspring of both control and diabetic pregnancy (r = -.74; P less than .02). Tissue cytochrome oxidase activity was lower in offspring of severely affected diabetic does, indicating a reduction in BAT mitochondrial content compared with controls. BAT mitochondria from fetuses of severely diabetic does exhibited reduced 3H-GDP capacity, which was 2.5-fold lower than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗

PubMed1992

Ca2+ potentiates corticotropin-induced, but not isoproterenol-induced, [3H]guanosine diphosphate release in rat adipocyte membranes.

EGTA abolished corticotropin (ACTH)-stimulated adenylate cyclase in rat adipocyte membranes. In contrast, the potency of guanosine triphosphate (GTP) stimulation of adenylate cyclase activated with ACTH was greater in the presence of Ca2+ (1 mmol/L). EGTA (1 mmol/L) powerfully inhibited ACTH-stimulated [3H]guanosine diphosphate (GDP) release from membranes prelabeled with [3H]GTP in the presence of isoproterenol (ISO) or ACTH, whereas Ca2+ significantly increased it. In contrast, neither EGTA nor Ca2+ affected ISO-stimulated [3H]GDP release. These data clearly show that Ca2+ is necessary for the binding of ACTH to its receptor, and that Ca2+ stimulates the interaction of the ACTH-occupied receptor with GTP-binding proteins.

Adenylyl Cyclases↗

PubMed1983

Angiogenic factors and their assay: activity of formyl methionyl leucyl phenylalanine, adenosine diphosphate, heparin, copper, and bovine endothelium stimulating factor.

A number of chemically unrelated substances have been compared for their neovasculogenic activity by two different in vivo tests, and for their ability to induce cultured endothelial cell migration and proliferation. Formyl methionyl leucyl phenylalanine, copper ions, heparin, adenosine diphosphate, and low-molecular-weight bovine endothelium stimulating factor were all neovasculogenic by the corneal pocket assay. By the chorioallantoic membrane assay, copper ions and formyl methionyl leucyl phenylalanine were not detectably neovasculogenic. By the same assay it has been possible to demonstrate angiogenic activity in bovine endothelium stimulating factor that is distinguishable from copper ions. This bovine factor, in contrast to the other agents, induced marked endothelial cell migration and also proliferation and may belong to a special class of agents which represents direct acting angiogenic activity.

Adenosine Diphosphate↗

PubMed1995

Structural changes induced by cytidine-5'-diphosphate choline (CDP-choline) chronic treatment in neurosecretory neurons of the supraoptic nucleus of aged CFW-mice.

The influence of chronic administration of cytidine-5'-diphosphate choline (CDP-choline), a precursor of the membrane lipid phosphatidylcholine, was studied in neurosecretory neurons (NSNs) of the supraoptic nucleus (SON) of aged mouse hypothalamus. Animals were treated with CDP-choline from 12 months of age until 26 months. They were studied for both morphologic and morphometric features. The results obtained were compared to those of an age-matched control group. There was evidence of differences between NSNs of the control group and those of the CDP-choline group which showed neuronal hypertrophy. This size increase was mainly attributed to the increment of cellular protein synthesis machinery, rough endoplasmic reticulum (RER) and Golgi complexes. Furthermore there was an increase in the number of neurosecretory granules (NSGs) in the CDP-choline group. In addition, there was no tertiary lysosomes in the treated animals. Moreover, the percentage of NSN membrane that was not covered by glial prolongations, increased from about 2% in the control group to 12% in the CDP-Choline treated group. These changes suggested an activation of the cellular processes for neurohormone synthesis in the experimental group. Furthermore, these NSNs displayed lipid droplets in their cytoplasm. The possible relationship between CDP-choline and NSNs activity is discussed.

Aging↗

PubMed1982

Platelet aggregation in laminar flow. I. Adenosine diphosphate concentration, time and shear rate dependence.

The relative contributions of physiologic activators and shear rates (G) on the formation and stability of platelet aggregates for in vitro laminar flow need to be characterized for conditions of low G believed to be important in vivo. Platelet aggregation was therefore studied in human citrated platelet-rich plasma (PRP) prepared at 37 degrees C for G = 0-190 sec-1 in laminar flow in a cone-in-plate device as a function of adenosine diphosphate (ADP) activation. Macroaggregation (TA) was assessed by visual observation and light transmission (%T), as routinely done in aggregometry, while actual percent aggregation (PA) was determined by microscopy of sub-samples from the decrease in single platelet concentration. Unactivated PRP never yielded shear-induced aggregation while samples pre-mixed with low [ADP] (less than or approximately 0.7 microM) led to large visible aggregate formation stable only for G less than or approximately 30-60 sec-1, apparently arising from pre-existing microaggregates. Direct ADP injections into the flow device showed that at low [ADP] (less than 1 microM), maximal values for both PA and TA were reached when step-wise increases in G reached less than or approximately 30 sec-1, with TA decreasing at higher G; these values were only some 10-20% of maximal values achieved with high (ADP] (greater than or approximately 4 microM), when PA was again maximal at G greater than or approximately 30 sec-1, but TA expressed as a rate of aggregation continued to increase with G up to 150 sec-1. The results are discussed in terms of the temporal shear-rate and ADP-dependent dynamics of a platelet stickiness factor, 's'.

Adenosine Diphosphate↗

PubMed1985

Possible participation of adenosine 5'-diphosphate, arachidonic acid and paf-acether in the platelet pro-aggregating effect of uncontrolled insulin-dependent diabetic erythrocytes in vitro.

The red blood cells (RBC) from uncontrolled insulin-dependent diabetics (U.IDD) induce aggregation of platelets from control subjects. This effect was not observed when using platelets made unsensitive to adenosine 5'-diphosphate (ADP), arachidonic acid (AA) or paf-acether. Since RBC from U.IDD are less deformable than those from control subjects, we treated normal RBC in vitro with glutaraldehyde. These rigid RBC were used to study aggregation of normal platelets and of those unsensitive to ADP, AA or paf-acether. Results obtained with glutaraldehyde-treated RBC were comparable to those obtained with RBC from U.IDD, i.e. aggregation was obtained with untreated platelets but not with platelets unsensitive to ADP, AA or paf-acether. It is hypothesized that aggregation of control platelets induced by RBC from U.IDD is purely mechanical at its origin but is ultimately mediated by ADP, AA or paf-acether.

Adenosine Diphosphate↗

PubMed1992

Low dose infusion of adenosine diphosphate prolongs bleeding time in rats and rabbits.

The effect of intravenous infusion of adenosine diphosphate (ADP) on haemostatic and thrombotic mechanisms was studied in rats and rabbits. Infusion of ADP (0.2-1 microMol/kg/min) in rabbits prolonged the skin capillary bleeding time threefold after between 1/2 and 2 hours of infusion. Prolongation of the bleeding time was parallelled by reduced in vitro sensitivity of platelets to ADP in citrated platelet-rich plasma. No major changes in respiratory frequency and heart rate were observed. On the other hand, infusion of adenosine (1 microMol/kg/min) did not affect the bleeding times. Intravenous ADP (0.1 microMol/kg/min) significantly prolonged the bleeding time in anaesthetized rats. Platelet and red cell counts before and during the ADP infusion indicated slight, reversible platelet aggregation. After 75 min the ratio between red cells and platelets resumed a steady pre-experimental level. Platinum wires placed in the abdominal aorta of rats to monitor thrombus formation during ADP infusion indicated an antithrombotic effect of prolonged, low dose infusion of ADP. Since infusion of adenosine did not affect the haemostatic mechanisms, these observations may indicate that ADP infusion desensitized the platelet responsiveness to aggregating stimuli in vivo, and/or that prostacyclin production by endothelium was stimulated by infusion of ADP.

Adenosine↗

PubMed1987

Inhibition of adenosine diphosphate-induced intravascular aggregation of rat platelets in vivo by 6-oxo-prostaglandin E1.

The antiaggregatory effects of 6-oxo-PGE1 were evaluated in vivo in the rat using a minimally invasive technique involving 111-Indium labelling of platelets. The antiaggregatory effects on adenosine diphosphate-induced aggregation were compared with those of prostacyclin (PGI2) and prostaglandin E1 (PGE1) following slow infusions and bolus injections. The rank order of antiaggregatory potency was PGI2 greater than 6-oxo-PGE1 greater than PGE1 while the rank order of duration of antiaggregatory effects was PGE1 greater than 6-oxo-PGE1 greater than PGI2. The kinetics of the antiaggregatory effects of these prostaglandins suggests that such actions are not mediated by direct effects on platelets, but through a secondary mechanism.

Adenosine Diphosphate↗

PubMed1990

A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase.

The product of the abnormal wing discs (awd) gene of Drosophila is 78% identical to the product of the nm23 gene of mammals, which is differentially expressed in certain metastatic tumors. We present evidence that the awd gene codes for a nucleoside diphosphate kinase (NDP kinase) and that this Awd/NDP kinase is microtubule associated. Neuroblasts in Drosophila larvae homozygous for a null mutation in the awd gene are arrested in metaphase, indicating that microtubule-associated Awd/NDP kinase plays a critical role in spindle microtubule polymerization.

Alleles↗

PubMed1980

Differential effects of local anesthetics and propranolol on arachidonic acid and adenosine 5'-diphosphate-induced aggregation in rabbit platelets.

The effects of several local anesthetics and propranolol, a beta receptor antagonist with local anesthetic activity, on adenosine 5'-diphosphate (ADP) and arachidonic acid (AA)-induced aggregation were studied in rabbit platelets. Platelet-rich plasma (PRP) was obtained from rabbits anesthetized with pentobarbital and changes in turbidity of PRP were measured with an aggregometer. Inhibition of platelet aggregation by the local anesthetics was expressed as the percent change in rate of aggregation from control value. Dibucaine was the most potent inhibitor of both AA- and ADP-induced aggregation. This inhibition was sensitive to external calcium concentration. In terms of potency dibucane > tetracine > lidocaine in inhibiting AA- and ADP-induced aggregation. Procaine was more effective against AA- than ADP-induced aggregation. Propanolol was comparable to the more potent local anesthetics. These data show that local anesthetics and propranolol inhibit both AA- and ADP-induced platelet aggregation and suggest that the mechanism of action is non-specific in nature.

Adenosine Diphosphate↗

PubMed1981

Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose-1,6-diphosphate.

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The Km for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a pKa of 6.5-6.7. The cooperative interaction of the multiple phosphoenolpyruvate (PEP) binding sites is independent of pH in the range of 5.7-7.8. Variation of the Vmax value with pH indicates the presence of a dissociated group (pKa 6.2-6.3) and of an undissociated group (pKa 7.5-7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S0.5 value for this substrate, as is an undissociated enzyme group with a pKa in the range 6.7-7.0. It is shown that PEP and frucotse-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme, FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.

Adenosine Diphosphate↗

PubMed1993

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Amino Acid Sequence↗

PubMed1995

Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate.

Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2'-azido-2'-deoxycytidine 5'-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.

Animals↗

PubMed1991

Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.

Amino Acids↗

PubMed1995

A third type of nucleoside diphosphate kinase from spinach leaves: purification, characterization and amino-acid sequence.

A third type of nucleoside diphosphate kinase (NDP kinase III), distinct from the previously described NDP kinases I and II (Nomura et al. (1991) Biochim. Biophys. Acta 1077, 47-55), was purified from spinach leaves to electrophoretic homogeneity. NDP kinase III was judged by SDS-PAGE and by gel filtration to have molecular masses of 17 kDa and 102 kDa, respectively, suggesting that it is composed of six subunits similarly to the other spinach isoforms, NDP kinases I and II. Amino-acid sequence analysis revealed the primary structure of NDP kinase III to be comprised of 153 amino-acid residues, the sequence of which exhibited 61% and 53% homology with those of NDP kinases I and II, respectively. In the reaction catalyzed by the three isoforms, the order of Km as phosphate acceptor was determined as GDP << ADP for NDP kinase III, different from those observed for NDP kinase I (ADP << GDP) and for NDP kinase II (GDP = ADP). These results suggest that the three isoforms may have distinct roles in regulating intracellular 5'-di- and 5'-triphosphonucleotide levels in spinach leaves.

Amino Acid Sequence↗