Uridine diphosphate D-glucose dehydrogenase of Aerobacter aerogenes

Uridine diphosphate d-glucose dehydrogenase (EC 1.1.1.22) from Aerobacter aerogenes has been partially purified and its properties have been investigated. The molecular weight of the enzyme is between 70,000 and 100,000. Uridine diphosphate d-glucose is a substrate; the diphosphoglucose derivatives of adenosine, cytidine, guanosine, and thymidine are not substrates. Nicotinamide adenine dinucleotide (NAD), but not nicotinamide adenine dinucleotide phosphate, is active as hydrogen acceptor. The pH optimum is between 9.4 and 9.7; the K(m) is 0.6 mm for uridine diphosphate d-glucose and 0.06 mm for NAD. Inhibition of the enzyme by uridine diphosphate d-xylose is noncooperative and of mixed type; the K(i) is 0.08 mm. Thus, uridine diphosphate d-glucose dehydrogenase from A. aerogenes differs from the enzyme from mammalian liver, higher plants, and Cryptococcus laurentii, in which uridine diphosphate d-xylose functions as a cooperative, allosteric feedback inhibitor.     

Adenosine Triphosphate Pools in Methanobacterium

Certain aspects of adenosine triphosphate (ATP) metabolism in the strict anaerobe Methanobacterium strain M.o.H. have been investigated. Results of growth yield studies suggest that ATP conservation is very inefficient (0.06 mole of ATP per mole of hydrogen) under the conditions used to grow the bacterium in a fermentor. Experiments designed to demonstrate net ATP formation in cell-free extracts were negative. In whole-cell studies, substances which decreased ATP pool levels and increased adenosine monophosphate (AMP) pool levels were air, chloroform, 2,4-dinitrophenol, carbonylcyanide-m-chlorophenylhydrazone, and pentachlorophenol. The results suggest that the latter compounds act either as inhibitors of electron transport or as uncouplers of an energy-linked process. All the above compounds also inhibit methane formation in cell-free extracts, an ATP-requiring process. Methods are described for estimation of ATP, adenosine diphosphate (ADP), and AMP in whole cells, with a sensitivity in the range of 10 to 200 pmoles. An apparatus for quick sampling from an anaerobic suspension of whole cells also is described.     

The levels of soluble nucleotides in wheat aleurone tissue

The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions.     

Chemotactic activity of extracellular nucleotideson human immune cells.

Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation.     

Poly(adenosine diphosphate ribose) glycohydrolase in Physarum polycephalum.

Poly(adenosine diphosphate ribose) glycohydrolase, which has thus far only been found in mammalian tissues, was found for the first time in the primitive eukaryotic slime mold Physarum polycephalum. The hydrolytic product of poly(adenosine diphosphate ribose) with this enzyme was identified as adenosine diphosphate ribose by paper and thin-layer chromatography. It is likely that the enzyme caused exoglycosidic hydrolysis. The optimal pH of this enzyme was 6.0, and the K_m value was 4.3 μm, as adenosine diphosphate ribose residues of polymer. Adenosine diphosphate ribose, ADP and ATP at a concentration of 0.1mm strongly inhibited the enzyme activity. 3′,5′-Cyclic AMP was inhibitory at a concentration of 1mm. The molecular weight of this enzyme was estimated to be 57,000.     

Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities

Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, when conventional methods of nucleotide extraction were employed. Under the reaction conditions specified in this report, the initial rate of hydrolysis of adenosine triphosphate was directly proportional to the activity of APase in the sample extracts and consequently can be used as a sensitive measure of APase activity. A method was devised for obtaining reliable nucleotide measurements in naturally occurring microbial populations containing elevated levels of APase activity. The metabolic significance of APase activity in microbial cells is discussed, and it is concluded that the occurrence and regulation of APase in nature is dependent upon microscale inorganic phosphate limitation of the autochthonous microbial communities.     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis

The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.     

Calcium-dependent release of adenosine and uridine nucleotides from A549 cells

Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15–20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34±13 nM ATP (5.6), 11±5 nM ADP (3.7), 3.3±1.2 nM AMP (1.4), 23±7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 μM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells.     

Purinergic signalling in the liver in health and disease

Purinergic signalling is involved in both the physiology and pathophysiology of the liver. Hepatocytes, Kupffer cells, vascular endothelial cells and smooth muscle cells, stellate cells and cholangiocytes all express purinoceptor subtypes activated by adenosine, adenosine 5′-triphosphate, adenosine diphosphate, uridine 5′-triphosphate or UDP. Purinoceptors mediate bile secretion, glycogen and lipid metabolism and indirectly release of insulin. Mechanical stress results in release of ATP from hepatocytes and Kupffer cells and ATP is also released as a cotransmitter with noradrenaline from sympathetic nerves supplying the liver. Ecto-nucleotidases play important roles in the signalling process. Changes in purinergic signalling occur in vascular injury, inflammation, insulin resistance, hepatic fibrosis, cirrhosis, diabetes, hepatitis, liver regeneration following injury or transplantation and cancer. Purinergic therapeutic strategies for the treatment of these pathologies are being explored.     

Glycogen in Methanothrix

An intracellular glycogen was purified and characterized from the acetoclastic bacteria Methanothrix str. FE, its average chain length was about 13 glucose residues. Acetyl-CoA was shown to be synthesized by the action of acetate thiokinase; in addition pyruvate synthase, phosphoenolpyruvate synthetase and enzymes of gluconeogenesis were detected in cell extracts. For glycogen synthase activity, both adenosine diphosphate glucose and uridine diphosphate glucose were used as glycosyl donors, apparent K _m were, respectively, 8 M for ADPGlc and 625 M for UDPGLe, at the opposite the V _m were the same for both precursors. This was in accordance with competition experiments and strongly suggested that only one glucosyl transferase was involved and that ADPGlc was the physiological glycosyl donor in Methanothrix str. FE. In addition branching enzyme activity (1-4-glucan-6-glucosyl transferase) was detected in cell extracts.Abbreviations ADPGlc adenosine diphosphate glucose - UDPGlc uridine diphosphate glucose     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

pS10147-2, a3.7 kb multicopy plasmid isolated from Streptomyces coelicolor

The following putative precursors of the pseudomurein were isolated from trichloroacetic acid extracts of Methanobacterium thermoautotrophicum: a uridine diphosphate activated derivative of glutamic acid and the uridine diphosphate activated peptides (see text). The activated glutamic acid residue and the three activated pepetides lack the glycan components N-acetylglucosamine and N-acetyltalosaminuronic acid present in the intact pseudomurein. In this case uridine diphosphate should be directly linked to the amino group of a glutamic acid residue, which represents a new mode of amino acid and peptide activation.     

Cold-Sensitive Mutant of Salmonella typhimurium Defective in Nucleosidediphosphokinase

A cold-sensitive mutant of Salmonella typhimurium defective in nucleosidediphosphokinase (ATP:nucleosidediphosphate phosphotransferase, EC 2.7.4.6) has been isolated and characterized. The mutant contains only 2% of the enzyme activity found in the parent, and the heat lability of this activity is 10 times that from the parent at 33 C. Mutant extracts lack the ability to convert any of 11 nucleoside diphosphates tested to the corresponding nucleoside triphosphates, but the nucleosidemonophosphatase activities are normal. Although the nucleoside triphosphate pools of the mutant are depressed significantly at the restrictive temperature (20 C), they are slightly elevated at the permissive temperature (37 C). The levels of guanosine pentaphosphate and guanosine tetraphosphate are dramatically increased. Two representative enzymes of pyrimidine de novo synthesis, aspartic transcarbamylase and dihydroorotate dehydrogenase, are fully repressed at both 37 and 20 C. Intracellular pools of uridine diphosphate are depressed at both permissive and restrictive temperature.     

Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N⁶-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge.     

Pyrophosphate and fructose 2,6-bisphosphate effects on glycolysis in pea seed extracts

The participation of pyrophosphate-dependent phosphofructokinase (PPi-PFK) in plant glycolysis was examined using extracts from pea seeds (Pisum sativum L. cv Alaska). Glycolysis starting with fructose 6-phosphate was measured under aerobic conditions as the accumulation of pyruvate. Pyruvate accumulated in a medium containing PPi and adenosine diphosphate at about two-thirds of the rate in a medium containing adenosine diphosphate and adenosine triphosphate (ATP). The PPi-dependent pyruvate accumulation had the same reactant requirements and sensitivity to glycolysis inhibitors, sodium fluoride, and iodoacetamide, as the well-established ATP-dependent glycolysis. Added fructose 2,6-bisphosphate stimulated both the PPi-dependent pyruvate accumulation and PPi-PFK activity whereas this modulator had no effect on ATP-dependent glycolysis or ATP-PFK. Collectively these results demonstrate a PPi-dependent glycolytic pathway in plants which is responsive to fructose 2,6-bisphosphate.     

Volume decrease of glycerinated polymorphonuclear leukocytes induced by ATP and Ca : 1. Resemblances to actomyosin contraction

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

Viability of cells reconstituted by virus-induced fusion of minicells with anucleate cells

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.