Plasma Membrane NADH Oxidoreductase Research Articles

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulindependent diabetes mellitns) patients and agematched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly reduced in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Multiple proteins with single activities or a single protein with multiple activities: The conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

VDAC1 Is a Transplasma Membrane NADH-Ferricyanide Reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Plasma membrane NADH‐oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (ρ0)

The mammalian plasma membrane (PM) NADH-oxidoreductase (PMOR) system is a multi-enzyme complex located in the plasma membrane of all eukaryotic cells, harboring at least two distinct activities, the plasma membrane NADH-ferricyanide reductase and the NADH-oxidase. To assess the behaviour of the two activities of the PMOR system, we measured the NADH-ferricyanide reductase and NADH-oxidase activities in fibroblast cell lines derived from patients carrying a mitochondrial DNA (mtDNA) G11778A mutation. We also measured the two activities in other cell lines, the HL-60 and HeLa (S3) lines, as well as in rho0 cells (cells devoid of mtDNA) generated from those lines and the fibroblast cells. These rho0 cells consequently lack oxidative phosphorylation and rely on anaerobic glycolysis for their ATP need. We have proposed that in rho0 cells, at least in part, up-regulation of the PMOR is a necessity to maintain the NAD+/NADH ratio, and a pre-requisite for cell growth and viability. We show here that the PM NADH-ferricyanide reductase activity was up-regulated in HL-AV2 (HL-60 rho0) cell lines, but not in the other rho0 and mtDNA mutant lines. The plasma membrane NADH oxidase activity was found to be up-regulated in both HL-AV2 and HeLa rho0 cell lines, but not significantly in the fibroblast rho0 and G11778A lines.

Plasma membrane NADH-oxidoreductase system: a critical review of the structural and functional data.

The observation in the early 1970s that ferricyanide can replace transferrin as a growth factor highlighted the major role plasma membrane proteins can play within a mammalian cell. Ferricyanide, being impermeant to the cell, was assumed to act at the level of the plasma membrane. Since that time, several enzymes isolated from the plasma membrane have been described, which, using NADH as the intracellular electron donor, are capable of reducing ferricyanide. However, their exact modes of action, and their physiological substrates and functions have not been solved to date. Numerous hypotheses have been proposed for the role of such redox enzymes within the plasma membrane. Examples include the regulation of cell signaling, cell growth, apoptosis, proton pumping, and ion channels. All of these roles may be a result of the function of these enzymes as cellular redox sensors. The emergence of many diverse roles for ferricyanide utilizing redox enzymes present in the plasma membrane might also, in part, be due to the numerous redox enzymes present within the membrane; the poor molecular characterization of the enzymes may be the reason for some of the diverging results reported in the literature as various researchers may be working on different enzymes. Here we review the diverse proposals given for structure and function to the plasma membrane NADH-oxidoreductase system(s) with a specific focus on those enzyme activities which can couple ferricyanide and NADH. Although they are still ill-defined enzymes, evidence is rising that they are of utmost significance for cellular regulation.

Glutamate neurotoxicity in rat cerebellar granule cells involves cytochrome c release from mitochondria and mitochondrial shuttle impairment.

To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell.

Apoptosis and redox homostasis: On a possible mechanism of action of Bcl-2

We discuss the involvement of several mammalian redox systems in the regulation of apoptosis. We focus especially on the role that mitochondria and the still ill-characterized plasma membrane NADH-oxidoreductase system play in apoptosis. The latter system was shown to respond to downregulation of mitochondrial function; inhibition of either system induces apoptosis. Apoptosis induced by inhibitors of the oxidase involves both Bcl-2 and calcineurin, two proteins recently shown to be capable of forming a tight complex. We suggest that Bcl-2 acts as an antioxidant, but in an electron sense rather than in an oxygen sense.

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators.

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specific in vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH:ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH:ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group of groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.

The universality of bioenergetic disease: The role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase

The universality of bioenergetic disease: The role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase

Analytical Subcellular Fractionation of Neutrophils from Patients with Chronic Granulomatous Disease<subtitle>DEMONSTRATION OF THE ENZYME DEFECT IN FOUR CASES</subtitle>

Analytical subcellular fractionation studies were performed on neutrophils from five patients, including two females, with chronic granulomatous disease. The density distribution and marker enzyme activities of the principal subcellular organelles in unstimulated cells were similar to those in unstimulated neutrophils from control subjects. NADH dependent reduction of nitroblue tetrazolium was measured in four of the patients including one female. In homogenates of whole cells the specific activity of this enzyme expressed as milliUnits/mg protein was lower in the patients than in the controls, but the difference was not statistically significant. There was however a highly significant difference between the specific activities of this enzyme in the plasma membrane fractions isolated from neutrophils of the four patients and the three controls. These findings suggest that the primary microbicidal oxidase of neutrophils, defective function of which manifests as the syndrome of chronic granulomatous disease, is a plasma membrane NADH oxidoreductase.