Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hydrogen peroxide (H2O2) is an essential electron acceptor for thyroid peroxidase-catalyzed iodination and coupling reactions. In the presence of iodide, its production is a limiting step in thyroid hormone biosynthesis. Several studies have demonstrated that the thyroid particulate fraction contains a Ca2+- and NADPH- dependent H@O@ generator (NADPH-O2:oxidoreductase), the so- called thyroid NADPH-oxidase. It has recently been demonstrated that cellular H2O2 release is under the tonic control of TSH in primary cultures of dog thyrocytes. The present study evaluates the effect of TSH on the thyroid NADPH-oxidase and cytochrome c reductase activities, two enzymes believed to be involved on H2O2 generation in the thyroid gland. There was almost no detectable NADPH-dependent H2O2 generator in the membranes of cells grown for 18 h without TSH. But cells grown in the presence of TSH (0.1 mU/ml) had a CA2+- and NADPH-dependent H2O2-generating activity that increased up to the third day in culture, as did the cell iodide organification capacity. This increase was also partially blocked by 12-O-tetradecanoylphorbol 13-acetate and cycloheximide. Forskolin and 8-bromo-cAMP both reproduced the action of TSH on the Ca2+- and NADPH-dependent H2O2 generator. In contrast, the thyroid NADPH-cytochrome c reductase activity in particles from control cells was similar to that of TSH-treated cells and was unaffected by forskolin or 12-O-tetradecanoylphorbol 13-acetate. These results suggest that NADPH-cytochrome c reductase activity is not regulated by TSH and, thus, reinforce the idea that this enzyme is not involved in thyroid H2O2 generation. On the other hand, the Ca2+- and NADPH-dependent H2O2 generator, so-called thyroid NADPH- oxidase, is induced by TSH through the cAMP cascade. Thus, it seems to be another marker of thyroid differentiation, in addition to thyroperoxidase and thyroglobulin, and could play a key role in thyroid hormone production.
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PMID:The Ca2+- and reduced nicotinamide adenine dinucleotide phosphate-dependent hydrogen peroxide generating system is induced by thyrotropin in porcine thyroid cells. 860 67

Iron catalyzed free radical formation and lipid peroxidation are accepted mechanisms of heme protein-induced acute renal failure. However, the source(s) of those free radicals which trigger lipid peroxidation in proximal tubular cells remains unknown. This study tested the potential involvement of mitochondrial electron transport, xanthine oxidase activity, and arachidonic acid metabolism in the heme-induced peroxidative state. The impact of cytosolic Ca2+ loading also was assessed. Rhabdomyolysis was induced in mice by glycerol injection, and two hours later heme-laden proximal tubular segments (PTS) were isolated for study. PTS from normal mice served as controls. During 30 to 60 minute incubations, heme loaded PTS developed progressive cytotoxicity (LDH release) and iron-dependent lipid peroxidation (malondialdehyde, MDA, generation; inhibited by deferoxamine). Site 2 (antimycin A) or site 3 (cyanide, hypoxia) mitochondrial respiratory chain inhibition completely blocked lipid peroxidation, whereas site 1 inhibition (rotenone) doubled its extent (presumably by shunting NADH through NADH dehydrogenase, a free radical generating system). Conversely, these agents did not substantially alter MDA in normal PTS. Normal and heme loaded PTS developed comparable degrees of LDH release during respiratory blockade irrespective of increased or decreased MDA production (indicating that lipid peroxidation was not a critical determinant of cell death). Neither increasing free arachidonic acid (PLA2 treatment) nor adding cyclooxygenase/lipoxygenase/cytochrome p450 inhibitors conferred a consistent protective effect. Altering free Ca2+ status (chelators; ionophore addition) and xanthine oxidase inhibition had no discernible impacts. Despite mitochondrial free radical production, mitochondrial function, as assessed by the ATP/ADP ratio, seemingly remained intact. In conclusion, (1) the terminal mitochondrial respiratory chain is the dominant source of free radicals which trigger PTS lipid peroxidation; (2) iron is a required secondary factor; (3) although mitochondria fuel lipid peroxidation, they do not appear to be critical targets of the heme-induced oxidant attack.
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PMID:Mitochondrial free radical production induces lipid peroxidation during myohemoglobinuria. 864 15

Heart mitochondria can be made to oxidize extramitochondrial NADH via the exogenous NADH dehydrogenase. Oxidation of extramitochondrial NADH was found to be associated with the disappearance of H+ from the suspension medium. Our studies on the possible pathway through which H+ may disappear from the extramitochondrial space were focused on (i) an unspecific transmembranous H+ leakage along the electrochemical H+ gradient following peroxidative membrane alteration, (ii) stimulation of a controlled H+ reconduction through the H+ channel of the ATP synthase, and (iii) stimulation of the Na+/H+ counterporter by Ca2+ release. Our experiments revealed that none of these H+ pathways was involved in the observed alkalinization of the extramitochondrial space during respiration of external NADH. The latter effect was inhibited when oxidation of external NADH via the respiratory chain was blocked and could be turned into the opposite when artificial e- acceptors of the exogenous NADH dehydrogenase were used to reactivate NADH consumption. Stoichiometric analysis of H+ disappearance and O2 consumption revealed that reducing equivalents of external NADH were transferred to oxygen via cytochrome oxidase and H+ from the suspension was used to release water.
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PMID:The effect of the exogenous NADH dehydrogenase of heart mitochondria on the transmembranous proton movement. 866 Jul 6

The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron (III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.
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PMID:Nature of inhibition of mitochondrial respiratory complex I by 6-Hydroxydopamine. 878 29

The distribution of the nicotinamide adenine dinucleotide phosphate-(NADPH) diaphorase reaction, an indicator of nitric oxide synthase activity, was studied in the freshwater planarian Dugesia tigrina (Platyhelminthes). The reaction was restricted to the pharynx, where the inner epithelium was intensely stained and the outer epithelium moderately stained. Neurons that innervated the pharynx were also stained. The enzyme activity was studied by high pressure liquid chromatographic quantitation of the formed citrulline. The presumed nitric oxide synthase was dependent on NADPH, whereas no dependency on Ca2+ and calmodulin could be detected. Tetrahydrobiopterin increased the activity about fivefold to 218.2+/-24.9 fmol/mg protein per min. Nomega-nitro-l-arginine depressed the enzyme activity by about 80%. The results indicate that nitric oxide has a role in the feeding behavior of planarians.
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PMID:Nitric oxide synthase in the pharynx of the planarian Dugesia tigrina. 892 43

The uptake and release properties of Ca2+ by several subcellular fractions of the bovine adrenal medulla were investigated. Investigation by the 45Ca2+ tracer method showed that permeabilized cells and the fractions of mitochondria (MT) and microsomes (MC) caused ATP-dependent Ca2+ uptake in a Ca2+ concentration-dependent manner (pCa 8-4), whereas permeabilized cells and the fractions of secretory granules (SG) were able to accumulate a significant amount of Ca2+ even in the absence of ATP, which was completed by the addition of hexokinase and glucose. In these organelle fractions, Ca2+ uptake in the presence of ATP at pCa 7 and pCa 5.8 was well-correlated with the activity of the NADPH cytochrome c reductase (marker enzyme for the endoplasmic reticulum) and cytochrome c oxidase (marker enzyme for mitochondria), respectively. As detected by Fura-2 ratiometry, both inositol 1,4,5-trisphosphate (IP3) and caffeine caused concentration-dependent Ca2+ releases from permeabilized cells and MC, but not from MT and SG. In an ATP-depleted condition, homogenates still took up a significant amount of Ca2+ but was not able to respond to IP3 and caffeine. These results suggest that the endoplasmic reticulum is a major Ca(2+)-storing organelle, which releases Ca2+ in response to IP3 and caffeine in bovine adrenal chromaffin cells.
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PMID:Inositol 1,4,5-trisphosphate- and caffeine-sensitive Ca(2+)-storing organelle in bovine adrenal chromaffin cells. 901 39

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM. The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1). Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.
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PMID:Characterization of human liver inducible nitric oxide synthase expressed in Escherichia coli. 922 37

The mechanism that underlies transcellular Ca2+ reabsorption in the kidney of the euryhaline teleost Oreochromis mossambicus was studied. Preparations of membrane vesicles made from the kidneys of freshwater- and seawater-adapted fish were more than sevenfold enriched in the basolateral plasma membrane marker Na+/K+-ATPase. Significant recovery of NADH­ cytochrome c reductase enzyme activity and of oxalate-stimulated Ca2+ pump activities in the membrane preparations indicated that the membrane fraction was of endoplasmic reticular origin. Indeed, thapsigargin specifically inhibited Ca2+ pump activity that could be attributed to oxalate-permeable endoplasmic reticular fragments. Kinetic analysis of thapsigargin-insensitive Ca2+ pump activity indicated the existence of a homogeneous, high-affinity, ATP-driven Ca2+ pump. No Na+-driven Ca2+ transport mechanism could be demonstrated. Plasma membrane Ca2+ pump activity was 56 % lower in preparations from seawater-adapted fish than in preparations from freshwater-adapted fish, suggesting a physiological role for this Ca2+ pump activity in renal Ca2+ handling by euryhaline species, with an involvement in the regulation of Ca2+ reabsorption.
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PMID:Calcium pump activities in the kidneys of Oreochromis mossambicus 931 41

Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.
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PMID:Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin. 933 25

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.
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PMID:Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. 935 14


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