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  • Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

The Journal of biological chemistry (2014-02-07)
Courtney E Sparacino-Watkins, Jesús Tejero, Bin Sun, Marc C Gauthier, John Thomas, Venkata Ragireddy, Bonnie A Merchant, Jun Wang, Ivan Azarov, Partha Basu, Mark T Gladwin
摘要

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

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钼, foil, thickness 0.1 mm, ≥99.9% trace metals basis
Sigma-Aldrich
钼, foil, thickness 0.025 mm, ≥99.9% trace metals basis
Sigma-Aldrich
钼, wire, diam. 1.0 mm, 99.95% trace metals basis
Sigma-Aldrich
钼, foil, thickness 1.0 mm, ≥99.9% trace metals basis
Sigma-Aldrich
Anti-MARC2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
钼, rod, 100mm, diameter 15.0mm, centerless ground, 99.9%
钼, foil, 150x150mm, thickness 0.20mm, annealed, 99.9%
钼, wire reel, 100m, diameter 0.025mm, hard, 99.95%
钼, wire reel, 10m, diameter 0.05mm, annealed, 99.95%
钼, foil, 6mm disks, thickness 0.5mm, annealed, 99.9%
钼, foil, 6mm disks, thickness 0.75mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.005mm, 99.9%
钼, foil, 8mm disks, thickness 0.006mm, 99.9%
钼, foil, 8mm disks, thickness 0.007mm, 99.9%
钼, foil, 8mm disks, thickness 0.008mm, 99.9%
钼, foil, 8mm disks, thickness 0.009mm, 99.9%
钼, foil, 8mm disks, thickness 0.0125mm, 99.9%
钼, foil, 8mm disks, thickness 0.015mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.015mm, as rolled, 99.9%
钼, foil, 8mm disks, thickness 0.01mm, 99.9%
钼, foil, 8mm disks, thickness 0.01mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.020mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.025mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.03mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.05mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.075mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.125mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.15mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.1mm, annealed, 99.9%
钼, foil, 8mm disks, thickness 0.20mm, annealed, 99.9%