Dissertationen zum Thema „Pyridine nucleotides“

Neutrophils (polymorphonuclear leukocytes) are short lived granulocytes that playa primordial role in host innate defense against invading pathogens. Freshly isolated neutrophils spontaneously undergo apoptosis when cultured, which is associated with oxidative stress. We found that there is a dramatic shift in the metabolism of the 5-lipoxygenase product 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) from its biologically inactive o-oxidation product in freshly isolated neutrophils to the potent granulocyte chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in neutrophils cultured for 24 h. o-oxidation of the chemoattractant leukotriene B4 (LTB4) was also reduced in aging neutrophils incubated with arachidonic acid, resulting in higher levels of LTB4. The reduced o-oxidation activity appeared to be due to a decrease in active LTB4 20-hydroxylase. In contrast, the increased 5-oxo-ETE formation was not associated with an increase in the amount of active 5-hydroxyeicosanoid dehydrogenase, which is required for its formation, but rather with a dramatic increase in its cofactor NADP +. NAD+ levels also increased, but NADPH levels remained unchanged after 24 h. There was also evidence for increased oxidative stress (high GSSG/GSH) in aging neutrophils. The changes in 5-HETE metabolism and pyridine nucleotides in cultured neutrophils could be inhibited by neutrophil survival factors and antioxidants. These results suggest that in severe inflammation, aging neutrophils that have evaded rapid uptake by macrophages may produce increased amounts of the chemoattractants 5-oxo-ETE and LTB4, resulting in delayed resolution of inflammation. Similarly, we found that the NADPH oxidase activator PMA caused a very rapid and dramatic increase in NADP + levels in both freshly isolated and cultured neutrophils, accompanied by a rapid increase in 5-oxo-ETE synthesis and reduced o-oxidation activity. Surprisingly, this was not accompanied by a corresponding decline in NADPH levels, which instead initially increased, but rather by a precipitous reduction in NAD+, which mirrored the increase in NADP+. These results suggest that the phosphorylation of NAD+ by NAD kinase may be very important for providing both NADP+ for 5-oxo-ETE synthesis and NADPH for the respiratory burst.

Šilumos taikymas įvairioms ligoms gydyti jau buvo naudojamas senovės Egipto, Graikijos, Romos civilizacijose daugiau kaip prieš 2000 m. pr. Šiuo metu hipertermija plačiai taikoma visame pasaulyje gydant vėžį, nes tai pigus ir patogus metodas turintis mažą šalutinį poveikį. Mokslininkai nustatė, kad vėžinės ląstelės greičiau žūsta esant aukštesnei už fiziologinę (41-45°C) temperatūrai, todėl hipertermija, derinama su kitais vėžio gydymo metodais (radioterapija, chemoterapija, imunoterapija ir chirurgija), tampa efektyvesniu metodu. Kadangi yra mažai žinoma apie hipertermijos poveikio mechanizmą sveiko audinio ląstelėms karščiavimo, hiperterminio vėžio gydymo ar gydymo termoabliacija metu, todėl yra svarbu nustatyti hipertermijos paveiktų ląstelių gyvybingumą bei hipertermijos poveikį adenino ir piridino nukleotidų koncentracijoms. Šio darbo tikslas buvo įvertinti hipertermijos, būdingos nutolusioms nuo termozondo audinio sritims poveikį, adenino ir piridino nukleotidų koncentracijoms žiurkės kepenų ląstelėse bei audinyje. Buvo naudojamas jonų porų efektyviosios skysčių chromatografijos metodas, leidžiantis vienoje chromatografinėje analizėje išskirstyti labai skirtingo hidrofobiškumo junginius. Taip pat buvo vertintas gyvų ir negyvų ląstelių skaičius gautoje hepatocitų suspensijoje panaudojant tripano mėlio metodą bei NAD(P)H fluorescencijos pokyčiai kepenų audinyje termoabliacijos metu. Gauti rezultatai parodė, kad išskirti hepatocitai pasižymėjo dideliu gyvybingumu (8... [toliau žr. visą tekstą]
The application of heat in the treatment of disease was first recorded in the ancient civilizations of Egypt, Greece, and Rome from as early as 2000 BC. Nowadays hiperthermia is widely using in cancer diseases in all the world. It was determined by many scientists that cancer cells are more sensitive for supraphysiological temperature (41-45°C) killing compared to normal cells. There are numerous evidences that hyperthermia can increase the effectiveness of other cancer therapies: radiotherapy, chemotherapy, immunotherapy and surgery. There is little known about the mechanisms of hyperthermia effects on healthy tissue, which are important in fever, in hyperthermic treatment of neighboring tumour and in thermoablation. Therefore it is very important to determinate the vability of cells during different hyperthermic treatment and hyperthermic effects of adenine ir pyridine nucleotides concentrations.The aim of study was to value the effect of hyperthermia,which is typical remote from thermoprobe tissue areas, on the concentration of adenine and pyridine nucleotides in hepatocytes and liver tissue. It was used ion-pair high-performance liquid chromatography method, which allows to disperse different combinations of hydrophobicity. Also were evaluated live and dead cells quantity in the suspension through tripan blue method and NAD(P)H fluorescence changes in liver tissue during the ablation. The results showed that isolated hepatocytes exhibited with high viability (80%)... [to full text]

L’infarctus du myocarde (IC) représente plus de 15 % de la mortalité mondiale liés aux maladies cardiovasculaires (MC). En absence d’une rapide reperfusion des coronaires occluses, aucune intervention thérapeutique n’est capable de limiter les effets délétères de l’IC. Un des moyens les plus efficaces de la cardioprotection, étudié en recherche, est le préconditionnement cardiaque ischémique (PCI). L’adénosine libérée au cours du PCI active via ces récepteurs P1 les voies de cardioprotection. Des études mettent en évidence également l’implication des purinorécepteurs P2Y dans la cardioprotection. Les récepteurs P2Y2, 4 et 6 sont les plus étudiés dans la cardioprotection. Les récepteurs P2Y sont sensibles aux nucléotides comme l’ATP et l’UTP libérés au cours de l’ischémie. Parmi les récepteurs P2Y seul le récepteur P2Y11 est doublement couplé à l’adénylate cyclase et à la phospholipase C (PLC). Il est également le seul récepteur dont le polymorphisme génétique, induisant une perte du signal métabotropique, prédispose à plus de risques d’IC et d’inflammation dans toutes les catégories d’âge indépendamment des facteurs de risques variables de l’IC (Amisten et al., 2007). Ceci suggère un important rôle du récepteur P2Y11 dans la prévention primaire et comme cible thérapeutique de l’infarctus du myocarde. Le β-NAD, nucléotide à base de pyridine, est libéré au cours de l’ischémie comme les médiateurs de cardioprotection notamment l’adénosine, l’ATP et l’UTP. Cependant, aucune étude ne s’est intéressée spécifiquement aux rôles du P2Y11 et des nucléotides pyridiniques, comme médiateurs, dans la cardioportection alors que le NAADP, un métabolite du β-NAD, est rapporté comme agoniste (Moreschi et al., 2008) du récepteur P2Y11. Dans ce travail, nous avons mis en évidence pour la première fois l’augmentation des concentrations interstitielles des métabolites du β-NAD comme le NAADP au cours de l’ischémie. L’augmentation des concentrations du NAADP décrit une cinétique comparable à celle de l’adénosine. Le NAADP extracellulaire ([NAADP]e), appliqué avant un cycle d’ischémie/reperfusion (I/R), déclenche une cardioprotection envers les effets délétères de l’I/R. En effet, le [NAADP]e améliore les fonctions contractiles, réduit les contractures, les arythmies de reperfusion et la taille de l’infarctus, dans un modèle d’I/R cardiaque chez le rat. Ce préconditionnement pharmacologique induit par le [NAADP]e implique les récepteurs P2Y11-like. Dans un modèle de cardiomyocytes, nous mettons en évidence l’activité métabotropique spécifique du récepteur P2Y11-like déclenché par le [NAADP]e. Le [NAADP]e déclenche via le récepteur P2Y11-like l’activation de la PKCε, ERK1/2, AKT et GSK-3β, des protéines kinases de prosurvie cellulaire, ce qui explique ses effets cardioprotecteurs. La phosphorylation des protéines de prosurvie cellulaire nécessite la médiation de la PKA, de la PLC et de la src. Le NF546, un nouvel agoniste sélectif du récepteur P2Y11, déclenche une activité métabotropique semblable à celle du [NAADP]e, confirmant ainsi l’existence fonctionnelle du récepteur P2Y11-like au niveau des cardiomyocytes de rat. L’activation du récepteur P2Y11-like, que ce soit par le [NAADP]e ou le NF546, induit une augmentation des concentrations intracellulaires du β-NAD et de ses métabolites, le NADP, le NAADP, le NAAD et l’ADP ribose cyclique. Le β-NAD, le NAADP comme le NAAD, ont été impliqués dans le déclenchement au niveau intracellulaire des voies de cardioprotection comme la voie des sirtuines et de l’autophagie. Ces mécanismes peuvent être complementaires de l’effet cardioprotecteur du [NAADP]e. L’ensemble de nos données conforte le rôle cardioprotecteur du récépteur P2Y11 et suggère que le NAADP interstitiel accumulé au cours de l’ischémie pourrait avoir un rôle de facteur paracrine de survie cellulaire améliorant la survie des cardiomyocytes au cours des évènements ischémiques
Myocardial infarction (MI) accounts for more than 15 % of global deaths related to cardiovascular diseases (CD). In the absence of a prompt reperfusion of occluded coronary arteries, none of therapeutic interventions is able to limit the deleterious effects of MI. One of the most effective means of cardioprotection, studied in research, is the ischemic cardiac preconditioning (ICP). Adenosine released during ICP triggers, via P1 receptors, cardioprotective effects. Involvement of the purinoceptors (P2Y) in cardioprotective effects has been also reported. P2Y2,4,6 receptors, the most studied P2Y receptors in cardioprotection, are activated by the nucleotides released during ischemia such as ATP and UTP. Among the P2Y receptors, P2Y11 is dually coupled to Gs and Gq proteins and is the single P2Y receptor which has been linked, via a genetic polymorphism, to an increased risk of acute myocardial infarction and elevated levels of C-reactive protein in humans in all age groups (Amisten et al., 2007). This suggests for the P2Y11 receptor an important role in primary prevention and as a therapeutic target of the P2Y11 receptor for myocardial infarction. β-NAD, a pyridine nucleotide, is released during ischemia like adenosine, ATP and UTP. However, no study has focused on the roles of P2Y11 receptor and pyridine nucleotide in mediating cardioprotective effects while NAADP, a β-NAD metabolite, has been reported as an agonist (Moreschi et al., 2008) of the P2Y11 receptor. In this work, we show, for the first time, increased interstitial concentrations of NAADP during ischemia. Interstitial kinetics of NAADP is similar to adenosine. Using a pharmacological preconditioning protocol, triggered by extracellular NAADP ([NAADP]e) before a prolonged ischemia/reperfusion (I/R), rat hearts rapidly recovered post-ischemic contractile function and displayed attenuated contracture, infarct size and arrhythmogenesis. This pharmacological preconditioning involves the P2Y11-like receptor. In cardiomyocytes culture, [NAADP]e induces specific metabotropic response of the P2Y11-like receptor. [NAADP]e triggers via the P2Y11-like receptor prosurvival protein kinases activation such as PKCε, ERK1/2, AKT and GSK-3β which explains its protective effects. Phosphorylation of prosurvival protein kinases requires the mediation of PKA, PLC and src. As with [NAADP]e, the NF546, a new selective agonist of P2Y11 receptor, triggers a metabotropic activity, thus confirming the functional existence of P2Y11-like receptor in rat cardiomyocytes. Activation of the P2Y11-like receptor, either by [NAADP]e or NF546 induced an increase in intracellular concentrations of β-NAD and its metabolites, NADP, NAADP, NAAD and cyclic ADP ribose. More, β-NAD, NAADP as well as NAAD have been involved in activation of cardioprotective pathways such as sirtuine pathways and autophagy. Taken together, our data demonstrate for the first time that the P2Y11 receptor mediates cardioprotective effects induced by [NAADP]e. NAADP is released during ischemia suggesting that [NAADP]e may act as a paracrine survival factor, prolonging cardiomyocytes lifespan during ischemic events

Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source. Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens. Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates. A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed. Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase. A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source.
Ph. D.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, three clones were isolated bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into pUC13. Examination of several deletion derivatives of the resulting plasmids and subsequent treatment with exonuclease BAL31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids and by in vitro transcription/translation of pDC21. Two polypeptides of molecular weights 52,000 and 48,000 were coded by the 3.05-kilobase fragment of pDC21. Both polypeptides were required for expression of transhydrogenase activity. The transhydrogenase was purified from cytoplasmic membranes of E. coli by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, α and β, of molecular weights 52,000 and 48,000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of fluorescence of 9-aminoacridine. It was concluded that E. coli transhydrogenase acts as a proton pump which is regulated primarily by a pH gradient rather than a membrane potential. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatorily linked. [¹⁴C]Dicyclohexylcarbodiimide preferentially labelled the a subunit. The transhydrogenase-catalyzed reduction of 3-acetylpyridine adenine dinucleotide by NADPH was stimulated over three-fold by NADH. It was concluded that NADH binds to an allosteric binding site on the enzyme. The nucleotide sequences of the pntA and pntB genes, coding for the transhydrogenase αand β subunits respectively, were established. The molecular masses of 53,906 (α) and 48,667 (β) and the N-terminal sequences of the predicted polypeptides agree well with the data obtained by analysis of the purified subunits. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed for each subunit.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.
Committee Chair: Marion B. Sewer; Committee Member: Alfred H. Merrill, Jr.; Committee Member: Donald F. Doyle; Committee Member: Dr. Edward T. Morgan; Committee Member: Kirill S. Lobachev. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The genes for the E. coli transhydrogenase enzyme have been cloned and sequenced in this lab and overexpressed in the membranes of E. coli (Clarke et al., 1986, Eur. J. Biochem. 158, 647-653). The E. coli transhydrogenase was found to consist of an α subunit (54588 Da) and a β subunit (48691 Da) arranged as an α₂β₂ dimer (Hou et al., 1990, Biochim. Biophys. Acta 1018, 61-66). The transhydrogenase enzyme was studied with respect to topology, location of the active sites, mechanism of proton pumping and mechanism of hydride transfer. The transhydrogenase was purified from E. coli membranes overexpressed with this enzyme as a soluble or a membrane-bound preparation. The soluble transhydrogenase was able to catalyze hydride transfer between ApNAD+ (3-acetylpyridine adenine dinucleotide) and NADPH, while in the membrane-bound transhydrogenase, this reaction was linked to the translocation of protons. The structure of the transhydrogenase was probed by limited trypsin digestion of both soluble and membrane-bound preparations. N-terminal amino acid sequences were obtained from the resulting fragments. These results led to a topological model of transhydrogenase in the membrane to be constructed. NADP+ and NADPH were found to introduce a conformational change in the β subunit resulting in two additional fragments derived from the β subunit upon trypsin digestion. Since transhydrogenase is known to contain separate binding sites for NAD(H) and NADP(H), the location of these was examined by covalent modification. FSBA (5'-pfluorosulfonylbenzoyladenosine) and DCCD (N,N'-dicyclohexylcarbodiimide) were both found to label near the NAD(H) binding site in the a subunit at aY226 and aD232,E238,E240 respectively. As well FSBA labelled another site in the p subunit at pY431, while DCCD labelled the transmembrane domain of the p subunit. The other site of FSBA labelling was proposed to be at the NADP(H) binding site. A residue βG314, when mutated, was found to abolish transhydrogenase catalytic activity as well as the NADP(H)-induced conformational change ability of the p subunit as probed by trypsin digestion. The sequence around this residue suggested the presence of another NADP(H) binding site on the p subunit. DCCD labelling followed by measurement of hydride transfer and proton translocation activities of wild-type transhydrogenase as well as a mutant where DCCD only labelled the transmembrane domain of the β subunit has shown that these two activities are coupled. The distance of DCCD labelling from the surface of the membrane was studied using NCD-4 (Ncyclohexyl- N'-[4-(dimethylamino)naphthyl]-carbodiimide), a fluorescent analog of DCCD, by quenching of the fluorescence with spin labels which intercalate into the membrane at various distances. The site of DCCD labelling in the transmembrane domain of the β has not been determined due to difficulty in isolating any sequencable peptide. Site-specific mutants of conserved residues in the transmembrane domains of the a and pp subunits were analyzed and PH91 was found to be implicated in proton translocation. A mutant, PH91N, demonstrated catalytic activity but this was not coupled to proton translocation activity. Therefore the two activities have become uncoupled in this mutant. The presence of two nucleotide binding sites on the β subunit in addition to the NAD(H) binding site on the α subunit was shown by affinity chromatography of the β subunit on NAD and NADP agarose columns, as well as by transhydrogenation between NADH and ApNAD+. In wild-type transhydrogenase, NADH reduced ApNAD+ only in the presence of NADP(H), but in mutants where the NADP(H)-induced cleavage of the p subunit had been disrupted so that p was cleaved in the absence of substrate, ApNAD+ was reduced by NADH in the absence of NADP(H). These experiments have demonstrated that there is a NAD(H) binding site on the a subunit and NADP(H) and NAD(H) binding sites on the β subunit and have given insight into the mechanism of hydride transfer.