Adenylate kinases of thermophiles Aquifex aeolicus and Geobacillus stearothermophilus: biochemical and kinetic studies

2021 ◽  
pp. 1-9
Author(s):  
Agnieszka Ludwiczak ◽  
Magdalena Wujak ◽  
Anna Kozakiewicz ◽  
Andrzej Wojtczak ◽  
Michał Komoszyński
Keyword(s):  
Atp Synthesis ◽  
Kinetic Studies ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Kinetic Properties ◽  
Geobacillus Stearothermophilus ◽  
Aquifex Aeolicus ◽  
Adenylate Kinases ◽  
Activity Of Enzymes ◽  
First Time

Adenylate kinases (AK) play a pivotal role in the regulation of cellular energy. The aim of our work was to achieve the overproduction and purification of AKs from two groups of bacteria and to determine, for the first time, the comprehensive biochemical and kinetic properties of adenylate kinase from Gram-negative Aquifex aeolicus (AKaq) and Gram-positive Geobacillus stearothermophilus (AKst). Therefore we determined KM and Vmax values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor Ap5A for both thermophilic AKs. The kinetic studies indicate that both AKs exhibit significantly higher affinity for substrates with the pyrophosphate group than for adenosine monophosphate. AK activation by Mg2+ and Mn2+ revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, Mn2+ ions at 0.2–2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than Mg2+ ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while Ap5A at a concentration of 10 µmol/L and 50 µmol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by Mg2+, suggesting the allosteric effect of ATP for both bacterial AKs.

Kinetic properties of glutamate dehydrogenase from the gills of Arapaima gigas and Osteoglossum bicirrhosum

1978 ◽  
Vol 56 (4) ◽  
pp. 809-813 ◽  
Author(s):  
J. H. A. Fields ◽  
W. R. Driedzic ◽  
C. J. French ◽  
P. W. Hochachka
Keyword(s):  
Glutamate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Energy Charge ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Nicotinamide Adenine ◽  
Kinetic Properties ◽  
Guanosine Diphosphate ◽  
Arapaima Gigas ◽  
Reduced Nicotinamide Adenine Dinucleotide

Glutamate dehydrogenase was isolated from the gills of Arapaima gigas and Osteoglossum bicirrhosum and kinetically characterized, in order to determine whether there was any alteration in the ability of the gills to generate ammonia associated with the development of an air-breathing life-style. The catalytic and regulatory properties of both enzymes were found to be very similar. They were activated by leucine, adenosine monophosphate, and adenosine diphosphate, and inhibited by guanosine triphosphate, guanosine diphosphate, and adenosine triphosphate. Inhibition by nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide was strong in both cases. It was concluded that both enzymes were regulated by a combination of the energy charge of the cell and the redox potential. There is no evidence for any qualitative alteration of the gills to produce ammonia from amino acids in the air breather, Arapaima gigas, as compared with the water breather, Osteoglossum bicirrhosum.

Mapping the Heart

2010 ◽  
Vol 18 (4) ◽  
pp. 6-8
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Receptor Subtypes ◽  
Cardiac Muscle Cells ◽  
Guanine Nucleotide Binding Protein ◽  
The Common ◽  
Guanine Nucleotide Binding ◽  
First Time ◽  
G Protein Coupled

Some of the receptors on the surface of cardiac muscle cells (cardiomyocytes) mediate the response of these cells to catecholamines by causing the production of the common second messenger cyclic adenosine monophosphate (cAMP). An example of such receptors are the β1- and β2-adrenergic receptors (βARs) that are heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. Selective stimulation of these two receptor subtypes leads to distinct physiological and pathophysiological responses, but their precise location on the surface of cardiomyocytes has not been correlated with these responses. In an ingenious combination of techniques, Viacheslav Nikolaev, Alexey Moshkov, Alexander Lyon, Michele Miragoli, Pavel Novak, Helen Paur, Martin Lohse, Yuri Korchev, Sian Harding, and Julia Gorelik have mapped the function of these receptors for the first time.

Selective sensing of adenosine monophosphate (AMP) over adenosine diphosphate (ADP), adenosine triphosphate (ATP), and inorganic phosphates with zinc(II)-dipicolylamine-containing gold nanoparticles

2021 ◽  
Author(s):  
Lena Reinke ◽  
Marcus Koch ◽  
Christine Müller-Renno ◽  
Stefan Kubik
Keyword(s):  
Gold Nanoparticles ◽  
Adenosine Triphosphate ◽  
Triethylene Glycol ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Mixed Monolayer ◽  
Inorganic Phosphates

Mixed monolayer-protected gold nanoparticles containing surface-bound triethylene glycol and dipicolylamine groups aggregated in water/methanol, 1:2 (v/v) in the presence of nucleotides, if the solution also contained zinc(II) nitrate to convert...

scholarly journals ON THE LOCALIZATION OF SOME PHOSPHATASES IN THREE DIFFERENT SEGMENTS OF THE PROXIMAL TUBULES IN THE RAT KIDNEY

1967 ◽  
Vol 15 (8) ◽  
pp. 456-469 ◽  
Author(s):  
N. O. JACOBSEN ◽  
F. JØRGENSEN ◽  
Å. C. THOMSEN
Keyword(s):  
Adenosine Triphosphate ◽  
Rat Kidney ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Proximal Tubules ◽  
Reaction Pattern ◽  
Cytoplasmic Bodies ◽  
Acid And Alkaline Phosphatase ◽  
Convoluted Tubules

The distribution of several phosphatases in three segments of the proximal tubules was studied in frozen sections of glutaraldehyde-fixed rat kidneys. Two segments of the convoluted tubules were identified by in vivo injection of trypan blue. By increasing the concentration of adenosine triphosphate to 3 mM in the Wachstein and Meisel ATPase medium, a clear segmental differentiation in the reaction pattern of the brush border, cytoplasmic bodies and basal infoldings of the proximal tubules was obtained. The specificity of the reaction was investigated by substituting adenosine diphosphate, adenosine monophosphate or β-glycerophosphate for adenosine triphosphate in the incubation medium and by employing cyanide or fluoride as inhibitors. The reaction pattern was also compared with the localization of acid and alkaline phosphatase activities. In addition, the distribution of glucose 6-phosphatase activity was studied which showed differences in the three segments of the proximal tubules.

Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of waveform and reversal of motion by calcium ions in vitro

1978 ◽  
Vol 33 (1) ◽  
pp. 235-253 ◽  
Author(s):  
J.S. Hyams ◽  
G.G. Borisy
Keyword(s):  
Calcium Ions ◽  
Beat Frequency ◽  
Flagellar Apparatus ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Living Cells ◽  
Exogenous Calcium ◽  
Ph Range ◽  
Relationship Of

The control of flagellar activity in the biflagellate green alga, Chlamydomonas reinhardtii was investigated by the in vitro reactivation of the isolated flagellar apparatus (the 2 flagella attached to their respective basal bodies plus accessory structures). The waveform and beat frequency of the isolated apparatus in the presence of 1 mM adenosine triphophate (ATP) were comparable to those recorded for living cells. Equimolar concentrations of adenosine diphosphate (ADP) could be substituted for ATP with little change in beat frequency and no apparent change in waveform, suggesting that the latter is converted to ATP by axonemal adenylate kinase. No reactivation occurred in adenosine monophosphate (AMP). But frequencies in cytidine, guanosine and uridine triphosphates (CTP, GTP and UTP) were approximately 10% that obtained in ATP. Reactivation was optimal over a broad pH range between pH 6.4 and pH 8.9 in both APT and ADP. Isolated flagellar apparatus could be induced to change from forward to reverse motion in vitro by manipulation of exogenous calcium ions. The 2 types of motion were directly comparable to recorded responses of living cells. Forward swimming occurred at levels of calcium below 10(−6)M, the isolated apparatus changing to backward motion above this level. Motility was inhibited at concentrations above 10(−3)M. The threshold for reversal of motion by calcium was lowered to 10(−7)M when the flagellar membranes were solubilized with detergent, indicating that the flagellar membranes are involved in the regulaion of the level of calcium within the axoneme. The reversal of motion by calcium was itself freely reversible. The relationship of these observations to the known tactic responses of Chlamydomonas is discussed.

scholarly journals Crowding within synaptic junctions influence the degradation of adenoside nucleotides by CD39 and CD73 ectonucleotidases

2021 ◽  
Author(s):  
Hadi Rahmaninejad ◽  
Tom Pace ◽  
Peter Kekenes-Huskey
Keyword(s):  
Electrostatic Interactions ◽  
Configurational Entropy ◽  
Reaction Diffusion ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Surprising Result ◽  
Surface Charges ◽  
Association Rate ◽  
Synaptic Junction ◽  
Reaction Diffusion Model

Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to a synaptic junction degradesuch nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine, oftentimes in a sequential manner. CD39 and CD73 are a representativeset of coupled ectonucleotidases, where CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which the AMP product is dephosphorylated into adenosine by CD73. Hence, CD39/CD73 help shape cellular responses to extracellular ATP. In a previous study [1] we demonstrated that the rates of coupled CD39/CD73 activity within synapse-like junctions are strongly controlled by the enzymes' co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within a synaptic junction, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39/CD73 electronucleotidase activity and in turn, the availability of intrasynapse ATP. Specifically, we simulated a spatially-explicit, reaction-diffusion model for the coupled conversion of ATP -> AMP and AMP -> adenosine in a model synaptic junction with crowders via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder/nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder/nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39/CD73 ectonucleotidase activity, electrostatics and crowding within synapses influence the availability of nucleotides for intercellular communication.

scholarly journals Studies on Human Erythrocyte Nucleotide Metabolism. II. Nonspherocytic Hemolytic Anemia, High Red Cell ATP, and Ribosephosphate Pyrophosphokinase (RPK, E.C. 2.7.6.1) Deficiency

Blood ◽  
1972 ◽  
Vol 39 (5) ◽  
pp. 674-684 ◽  
Author(s):  
William N. Valentine ◽  
Helen M. Anderson ◽  
Donald E. Paglia ◽  
Ernst R. Jaffé ◽  
Patricia N. Konrad ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Reduced Glutathione ◽  
Adenine Nucleotides ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Blood Film ◽  
Nucleotide Metabolism ◽  
Glutathione Metabolism ◽  
Final Decision ◽  
Blood Samples

Abstract A 29-yr-old black woman was found to have a long-standing, nonspherocytic hemolytic disorder associated with a marked reduction in the activity of erythrocyte ribosephosphate pyrophosphokinase (RPK, PRPP synthetase, E.C. 2.7.6.1). Although the patient’s erythrocytes had about 50% of the average RPK activity of normal mature human erythrocytes, this level represented only about 20-30% of the activity in comparable reticulocyte-rich blood samples from patients with other types of hemolytic anemias. The concentrations of adenosine triphosphate adenosine diphosphate, adenosine monophosphate and, therefore, of total adenine nucleotides in her erythrocytes were markedly increased, even well above the levels in extracts of comparable reticulocyte-rich blood samples. ATPase activity was increased three- to fourfold, consistent with the reticulocytosis. Adenylate kinase and adenine phosphoribosyltransferase activities were normal. The activities of all enzymes of the Embden-Meyerhof and hexose monophosphate shunt pathways and enzymes related to glutathione metabolism were normal or increased, consistent with the reticulocytosis. The concentrations of glycolytic intermediates, other than adenine nucleotides, were normal. The conversion of glucose, adenosine, and inosine to lactate was normal or increased. Autohemolysis was of the Dacie Type II. The concentrations of erythrocyte-reduced glutathione were high normal or elevated. The stained blood film showed a striking degree of basophilic stippling of the erythrocytes. Studies of the erythrocytes of the patient’s only known relative, a son, have failed to reveal any hematologic or enzymatic abnormalities. A direct causal relationship between RPK deficiency, high ATP concentrations, and nonspherocytic hemolytic anemia could not be derived from data now available. The final decision as to whether the deficiency is primary and causative or is an epiphenomenon requires investigation of additional cases.

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