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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 (A[K.sub.aq]) and Gram-positive Geobacillus stearothermophilus (A[K.sub.st]). Therefore we determined [K.sub.M] and [V.sub.max] values, and the effects of temperature, pH, metal ions, donors of the phosphate groups and inhibitor A[p.sub.5]A 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 [Mg.sup.2+] and [Mn.sup.(2)+] revealed that both ions are efficient in the synthesis of adenosine diphosphate and adenosine triphosphate; however, [Mn.sub.(2)+] ions at 0.2-2.0 mmol/L concentration were more efficient in the activation of the ATP synthesis than [Mg.sup.2+] ions. Our research demonstrates that zinc ions inhibit the activity of enzymes in both directions, while A[p.sub.5]Aat a concentration of 10 [micro]mol/L and 50 [micro]mol/L inhibited both enzymes with a different efficiency. Sigmoid-like kinetics were detected at high ATP concentrations not balanced by [Mg.sup.(2)+], suggesting the allosteric effect of ATP for both bacterial AKs. Key words: adenylate kinase (AK), kinetic studies, nucleotide, Aquifex aeolicus, Geobacillus stearothermophilus. Les adenylate kinases (AK) jouent un role essentiel dans la regulation de l'energie cellulaire. L'objectif de ces travaux etait de parvenir a la surproduction et a la purification des AK de deux groupes de bacteries et, pour la premiere fois, de determiner les proprietes biochimiques et cinetiques completes de l'adenylate kinase d'Aquifex aeolicus (A[K.sub.aq])a Gram negatif et de Geobacillus stearothermophilus (A[K.sub.st]) a Gram positif. Les auteurs ont donc determine les valeurs de [K.sub.M] et de [V.sub.max], ainsi que les effets de la temperature, du pH, des ions metalliques, des donneurs de groupes phosphate et de l'inhibiteur A[p.sub.5]A pour les deux AK thermophiles. Les etudes cinetiques indiquent que les deux AKs presentent une affinite significativement plus elevee pour le groupe pyrophosphate que pour l'AMP en tant que substrats. L'activation des AK par le [Mg.sup.2+] et le [Mn.sup.(2)+] revele que les deux ions sont efficaces dans la synthese de l'ADP et de l'ATP; toutefois, les ions [Mn.sup.(2)+] aune concentration de 0,2-2,0 mmol/Letaient plus efficaces dans l'activation de la syn-thesedel'ATP que les ions [Mg.sup.(2)+]. Ces recherches demontrent que les ions zinc inhibent l'activite des enzymes dans les deux directions, tandis que l'A[p.sub.5]A a une concentration de 10 [micro]mol/L et 50 [micro]mol/L inhibait les deux enzymes avec une efficacite differente. Une cinetique de type sigmoide a ete detectee a des concentrations elevees d'ATP non equilibrees par le [Mg.sup.(2)+], ce qui suggere un effet allosterique de l'ATP pour les deux AK bacteriennes. [Traduit par la Redaction] Mots-cles: adenylate kinase (AK), etudes cinetiques, nucleotide, Aquifex aeolicus, Geobacillus stearothermophilus., Introduction Adenylate kinases (AKs, EC 2.7.4.3) are low-molecular-weight phosphotransferases (21-25 kDa) catalysing transphosphorylation reactions according to the equations (Krishnamurthy 2005): [Mg.sup.(2+]) : ATP + AMP [right arrow] [Mg.sup.2+] : ADP [...]

Purinergic signaling, the oldest evolutionary transmitter system, has been increasingly studied as a pivotal target for novel anti‐cancer therapies. In the present work, the developed nanobiocatalytic system consisting of adenylate kinase immobilized on graphene oxide (AK‐GO) was characterized in terms of its physicochemical and biochemical properties. We put special emphasis on the AK‐GO influence on purinergic signaling components, that is, ecto‐nucleotides concentration and ecto‐enzymes expression and activity in human lung carcinoma epithelial (A549) cells. The immobilization‐dependent modification of AK kinetic parameters allowed for the removal of ATP excess while maintaining low ATP concentrations, efficient decrease in adenosine concentration, and control of the nucleotide balance in carcinoma cells. The cyto‐ and hemocompatibility of developed AK‐GO nanobiocatalytic system indicates that it can be successfully harnessed for biomedical applications. In A549 cells treated with AK‐GO nanobiocatalytic system, the significantly decreased adenosinergic signaling results in reduction of the proliferation and migration capability of cancer cells. This finding is particularly relevant in regard to AK‐GO prospective anti‐cancer applications. [ABSTRACT FROM AUTHOR]

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1) sputum samples (healthy, n = 4; COPD, n = 37), 2) lung tissue samples (healthy, n = 13; smokers without COPD, n = 10; smoker+COPD, n = 17), 3) pulmonary lobectomy tissue samples (no/mild emphysema, n = 6), and 4) blood samples (healthy, n = 6; COPD, n = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)–resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1–mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers. [ABSTRACT FROM AUTHOR]

Extracellular purines, principally adenosine triphosphate and adenosine, are among the oldest evolutionary and widespread chemical messengers. The integrative view of purinergic signaling as a multistage coordinated cascade involves the participation of nucleotides/nucleosides, their receptors, enzymes metabolizing extracellular nucleosides and nucleotides as well as several membrane transporters taking part in the release and/or uptake of these molecules. In view of the emerging data, it is evident and widely accepted that an extensive network of diverse enzymatic activities exists in the extracellular space. The enzymes regulate the availability of nucleotide and adenosine receptor agonists, and consequently, the course of signaling events. The current data indicate that mesenchymal stem cells (MSCs) and cells induced to differentiate exhibit different sensitivity to purinergic ligands as well as a distinct activity and expression profiles of ectonucleotidases than mature cells. In the proposed review, we postulate for a critical role of these enzymatic players which, by orchestrating a fine‐tune regulation of nucleotides concentrations, are integrally involved in modulation and diversification of purinergic signals. This specific hallmark of the MSC purinome should be linked with cell‐specific biological potential and capacity for tissue regeneration. We anticipate this publication to be a starting point for scientific discussion and novel approach to the in vitro and in vivo regulation of the MSC properties. Mesenchymal stem cells (MSCs) and cells induced to differentiate exhibit different sensitivity to purinergic ligands as well as a distinct activity and expression profiles of ectonucleotidases than mature cells. The enzymes regulate the availability of nucleotide and adenosine receptor agonists, and consequently, the course of signaling events. This specific hallmark of the MSC purinome should be linked with cell‐specific biological potential and capacity for tissue regeneration. [ABSTRACT FROM AUTHOR]

Adenylate kinase (AK, EC 2.7.4.3) is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy β – and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd) and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD)) link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase. [ABSTRACT FROM AUTHOR]

This publication presents results of the recent studies on plant NTPDases (apyrases). The structure and major physicochemical properties of this enzymes are reviewed. The attention has been paid to metabolic functions of apyrases from Solanum tuberosum and Arabidopsis thaliana. Apyrases constitute a family of proteins hydrolyzing phosphoanhydride bonds of nucleoside tri- and di-phosphates. They share common features like a similar structure, broad nucleotide substrate specificity and divalent cation requirement for their catalytic activity. The presence of plant NTPDases was detected in various cellular compartments. They are soluble or membrane-bound proteins. In hydrolytic processes catalyzed by activity of apoplastic apyrases and other ectoenzymes, adenine, ribose and orthophosphate are produced. These compounds are transported to the cell. Apyrases have been speculated to be involved in the regulation of starch synthesis and signal transmission. Their activity is necessary for development and growth of tubers and roots. Enzymes from leguminous plants activate the symbiosis with root nodule bacteria. Considering the fact, that NTPDases change the nucleotide concentration in cells and tissues, most of described functions may be related to the regulation of the energy charge of cell.