26 research outputs found

    Nitric Oxide Synthase Encapsulation in Liposomes: A Potential Delivery Platform to (Nitric Oxide)-Deficient Targets

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    Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes: such as neuronal signaling, immune response and inflammatory response. In mammalian organisms, NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. Typically, large NO fluctuations in biological systems under/over a critical limit is associated with problems that range from transient dysfunctions to severe chronic disease states. In this regard, we explore the development of a potential delivery and release method of nitric oxide to NO-deficient sites using liposomes as vehicles. Liposomes have already been used as effective nano-carriers. In this short communication, we report on the preparation and characterization of liposomes carrying a recombinant NOS enzyme. We report on the efficacy of using liposomes to carry NOS enzymes, and on the extent of preservation of native NOS structure and function. In addition to the characterization of liposome stability and recovery of enzymatic activity after encapsulation in liposomes, we also measured the NO production upon NOS stimulation. The NO release was monitored with a nitric oxide ultrasensitive electrochemical microsensor placed near NOS-carrying liposomes. This method of NOS-carrying liposomes shows the promise of potential development as a platform for targeted NO-delivery

    Endothelial Nitric Oxide Synthase Oxygenase on Lipid Nanodiscs: A Nano-Assembly Reflecting Native-Like Function of eNOS

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    © 2017 Elsevier Inc. Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∌65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain

    Distinct Conformational Behaviors of Four Mammalian Dual-Flavin Reductases (Cytochrome P450 Reductase, Methionine Synthase Reductase, Neuronal Nitric Oxide Synthase, Endothelial Nitric Oxide Synthase) Determine Their Unique Catalytic Profiles

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    Multidomain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions and relationships between these parameters and catalytic activity are not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian diflavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase, methionine synthase reductase and endothelial and neuronal nitric oxide synthase. We used stopped-flow spectroscopy, single turnover methods and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. The results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12% to 72% open state) and also vary 100-fold with respect to their rates of conformational switching between unreactive closed and reactive open states (cytochrome P450 reductase \u3e neuronal nitric oxide synthase \u3e methionine synthase reductase \u3e endothelial nitric oxide synthase). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. The present study is the first to quantify these conformational parameters among the diflavin enzymes and suggests how the parameters might be manipulated to speed or slow biological electron flux

    Nitrite Biosensing via Selective Enzymes—A Long but Promising Route

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    The last decades have witnessed a steady increase of the social and political awareness for the need of monitoring and controlling environmental and industrial processes. In the case of nitrite ion, due to its potential toxicity for human health, the European Union has recently implemented a number of rules to restrict its level in drinking waters and food products. Although several analytical protocols have been proposed for nitrite quantification, none of them enable a reliable and quick analysis of complex samples. An alternative approach relies on the construction of biosensing devices using stable enzymes, with both high activity and specificity for nitrite. In this paper we review the current state-of-the-art in the field of electrochemical and optical biosensors using nitrite reducing enzymes as biorecognition elements and discuss the opportunities and challenges in this emerging market

    Nitric Oxide Synthase Encapsulation in Liposomes: A Potential Delivery Platform to (Nitric Oxide)-Deficient Targets

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    Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes: such as neuronal signaling, immune response and inflammatory response. In mammalian organisms, NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. Typically, large NO fluctuations in biological systems under/over a critical limit is associated with problems that range from transient dysfunctions to severe chronic disease states. In this regard, we explore the development of a potential delivery and release method of nitric oxide to NO-deficient sites using liposomes as vehicles. Liposomes have already been used as effective nano-carriers. In this short communication, we report on the preparation and characterization of liposomes carrying a recombinant NOS enzyme. We report on the efficacy of using liposomes to carry NOS enzymes, and on the extent of preservation of native NOS structure and function. In addition to the characterization of liposome stability and recovery of enzymatic activity after encapsulation in liposomes, we also measured the NO production upon NOS stimulation. The NO release was monitored with a nitric oxide ultrasensitive electrochemical microsensor placed near NOS-carrying liposomes. This method of NOS-carrying liposomes shows the promise of potential development as a platform for targeted NO-delivery

    Peroxynitrite and Nitroxidative Stress: Detection Probes and Micro-Sensors. A Case of A Nanostructured Catalytic Film

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    Peroxynitrite, the primary product of the reaction of superoxide ion and nitric oxide, emerged as an important species with profound biological roles. Relatively speaking, it is a new member of the nitroxidative array of reactive metabolites, and details of its actions, impact on biological systems in health and disease states are still accumulating. It has already been linked to a host of pathological conditions. At the same time, its cytoprotective roles including redox regulation of critical signaling pathways are also reported. Assessment of peroxynitrite’s deleterious versus potential protective/signaling roles strongly depends on the possibility to accurately measure and monitor its concentration. This will help build a clearer understanding of its physiological roles. However, peroxynitrite’s extremely short half-life under physiological conditions and its very complex reactivity with many cellular targets create a major analytical chemistry challenge, particularly at the single cell level. The dynamic concentration of peroxynitrite versus other reactive species generated in situ under various conditions modulates its role in many vital cell functions. In this chapter, we give a brief overview of peroxynitrite biochemistry, physiology, and related therapeutic efforts to control its impact under pathological conditions. We then discuss the challenges and accomplishments in terms of major analytical methods developed for peroxynitrite’s sensing up-to-date, as well as opportunities for the future
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