Peroxidase, Glutathione

For enzyme extracts and assays, fresh roots (0.1 g) were ground in liquid nitrogen, and then suspended in 0.9 mL solution containing 10 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 4°C, 2500 rpm for 10 min and the resulting supernatant was collected for determination of the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and glutathione peroxidase (GSH-Px, EC 1.11.1.9) using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All enzymes above were detected using a microplate reader (SpectraMax M5, USA), and 5 to 10 seedlings were used to provide enough amounts of root tissues in each experimental replicate (n = 3).
The activity of SOD was determined by measuring the inhibiting rate of the enzyme to O₂⁻· produced by the xanthine morpholine with xanthine oxidase using the SOD assay kit. Each endpoint assay was detected the red substances of the reaction system by absorbance at 550 nm after 40 min of reaction time at 37°C. And one unit SOD activity (U) was defined as the quantity of SOD required to produce 50% inhibition of reduction of nitrite in 1 mL reaction solution by measuring the change of absorbance at 550 nm.
The CAT activity was measured based on the hydrolysis reaction of hydrogen peroxide (H₂O₂) with CAT, which could be terminated by molybdenum acid (MA) to produce yellow MA-H₂O₂ complex. CAT activity was calculated by the decrease in absorbance at 405 nm due to the degradation of H₂O₂, and one unit is defined as the amount of enzyme that will cause the decompose of 1 µmol hydrogen peroxide (H₂O₂) per second at 37°C in 1.0 g fresh tissue according to CAT assay kit.
The POD activity was measured based on the change of absorbance at 420 nm by catalyzing H₂O₂. One unit was defined as the amount of enzyme which was catalyzed and generated 1 µg substrate by 1.0 g fresh tissues in the reaction system at 37°C. POD activity was calculated as the formula according to POD assay kit.
The GSH-Px activity was also measured using the assay kit based on the principle that oxidation of glutathione (GSH) and hydrogen peroxide (H₂O₂) could be catalyzed by GSH-Px to produce oxidized glutathione (GSSG) and H₂O. In addition GSH reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) to produce stable yellow substances and the decrease of GSH at 412 nm during the reaction is indicative of GSH-Px activity in tissues. One GSH-Px unit of GSH-Px activity (U) was calculated as the amounts of enzyme that will oxidize 1 µmol/L GSH in reaction system at 37°C per minute in 1.0 g fresh tissue according to the assay kit. All of the enzymes were expressed as in U/g FW.

Flies.
Drosophila wild-type strains Dahomey (DAH),
Canton S (CS) and Oregon R (OR) were obtained from stock-centers or
collaborators. The dj-1β^GE23381 mutant [31 (link)]: and
AOX-transgenic lines F6 and F24 [30 (link)] were as described previously. Flies were
maintained in a standard medium [30 (link)], collected using CO₂ anesthesia
within 24 h of eclosion, and then kept at a density of 20 flies per vial at 25
ºC in a controlled 12 h light:-dark cycle. Vials were changed every 2-3 days.
We have created two new wild type strains of Drosophilamelanogaster backcrossing for eleven generations DAH virgin females with OR males and OR
virgin females with DAH males. The new strains of Drosophila melanogaster are
called DAHOR (flies with nuclear DAH DNA and mitochondrial OR DNA) and ORDAH
(flies with nuclear OR DNA and mitochondrial DAH DNA).


Lifespan studies.
Between
180 and 400 flies were used for each study. Each independent study was repeated
twice: data were pooled and analysed together. Flies were collected within 24 h
after eclosion using CO₂ anaesthesia and kept at a density of 20
flies per vial at 25 ºC in a controlled 12 h light:-dark cycle.
Every 2-3 days vials were changed and the number of dead flies was counted,
from which mean and maximum lifespan (MLS, the last 10% of surviving flies)
were calculated. Prism GraphPad software was utilized to build survival curves
that were further analysed using the Kaplan Meier Log-Rank Test.


Mitochondrial
biochemistry.
Mitochondria were isolated according to Miwa et al.[31 (link)] with some minor modifications [30 (link)]. Mitochondrial respiration rates
were measured by polarography using a Clark-type oxygen electrode as previously
[30 (link)], in the absence or presence of KCN (100 μM), antimycin
A (10 μM) or rotenone (5 μM). Mitochondrial
ROS production was assayed according to the method described by [32 ] adapted to
flies [30 (link)].


RNA
quantification.
Total RNA was extracted from 10 days old flies
according to [30 (link)]. For cDNA synthesis, 13
μl reaction mixes containing 2 μg RNA, 1 μl DEPC 10 mM dNTP mix (Fermentas),
0.4μl Random Primers (0.5ug/μl Promega) and DEPC-treated water were
incubated at 90°C for 3 min, then transferred to ice, where 4 μl 5x M-MuLV
reaction buffer (Fermentas) and 1μl 40U/μl RNase inhibitor
(Fermentas) were added. The reactions were mixed and incubated at 25°C for 10
min. On ice, 2μl of 20U/μl M-MuLV reverse transcriptase (Fermentas)
was added, and the reaction was incubated for a further 10 min at 25°C, 1 h at
37°C and 70°C for 10 min. mRNA levels were analyzed by Q-RT-PCR. The transcript
levels of RpL32, Catalase, Superoxide dismutase 1 and 2 and Glutathione
Peroxidase were measured using primers pairs shown in supplementary Table 2. All RNA extractions were performed in triplicate, with each used as a
template for three separate cDNA synthesis reactions which were then pooled.
Each cDNA pool was itself analysed in triplicate. Expression of the target
genes was measured relative to that of RpL32 (rp49), in order to
normalize for sample and run to run variations. A series of 10-fold dilutions
of an external standard was used in each run to produce a standard curve.
Analytical reactions were performed using 20-fold diluted cDNA samples, in 25
μl reaction volume consisting of 2 μl of the cDNA template,
0.4μl of 20 μM forward and reverse primers, and 12.5ul of 2x MAXIMA
SYBR GREEN Master Mix (Fermentas). The PCR program consisted of a 10 min
pre-incubation at 95°C, 40 cycles of 35 secs denaturation at 95°C, 30 secs
annealing at 60°C and 30 secs extension at 72°C. Melting curve analysis,
consisting of a 15 secs denaturation step at 95°c followed by a 1 min
annealing step at 60°C and a 0.3°C/s denaturation ramp to 95 °C, was performed
after the amplification step to verify that only a single, specific extension
product had been amplified. Data were extracted and analysed using Applied
Biosystems StepOne software version 2.0.


Resistance to inhibitors of the ETC.
To
check the expression and activity of AOX in vivo experiments with a
variety of ETC inhibitors were performed. 20 flies
were kept (males and females separately) in fresh vials. To measure resistance
to KCN, the drug was dissolved in water at varying concentrations and added
directly to the food vial. Resistance to antimycin and rotenone was assayed
essential as described by Fridell et al. [33 (link)]. In brief, 2-3 day old flies were
starved for two hours in empty vials, following this flies were placed in vials
containing Whatman paper (3 mm x 1 mm) impreg-nated with 5% (w/v) sucrose
solution and the appropriate drug (3 mM antimycin or rotenone). Under these
conditions without any drug, flies are able to survive more than 72 h so any
effect before this time should be considered to be provoked by exposure to the
drug. The proportion of flies surviving was recorded over 24 h.


Sequencing
of Mitochondrial gene cytochrome c oxidase subunit I.
Mitochondrial
DNA was extracted using standard procedures from mitochondria isolated from
around 150 flies according to Miwa et al. [31 (link)] High fidelity PCR using
specific primers CoIF2 and CoIR5 (Supplementary Table 2) were used to amplify a 2.6 kb
fragment containing the cytochrome c oxidase subunit I (CG34067, CoI). PCR
products were purified using a Machary - Nagel PCR purification kit according
to manufacturer's instructions. Products were sequenced using Big dye
Terminator Chemistry 3.1v (Applied Biosystems) and a 3130 AB genetic analyser.
AB sequencing analysis software was used for analysis of electropherograms.


Statistical analysis.
Data were
analysed using GraphPad Prism 4 and one-way ANOVA was used for statistical
testing. When ANOVA was significant (p < 0.05) Newman-Keuls Multiple
Comparison test was also used. Lifespan data were analysed using the Kaplan
Meier Log-Rank Test. The statistically significant value was established as p < 0.05.

The superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activity was evaluated as already described previously [47 ,48 (link)]. SOD activity was evaluated by quantitative sandwich enzyme immunoassay technique using a commercial kit (Cusabio, Houston, Texas). The results were expressed as U/mg of protein. For GPx activity, the absorbance was monitored at 340 nm at 37 °C for 10 min, and the results were expressed as μmol of reduced glutathione (GSH)/min/mg of protein. The GR activity was evaluated by measuring the consumption of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor in the reduction of oxidized glutathione to reduce GSH. The results were expressed as U of GR/mg of protein. One U of enzyme activity was defined as the amount of GR that oxidizes 1 μmol of NADPH per min. The GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The results were expressed as U of GST/mg of protein. One U of enzyme activity was defined as the amount of GST that produces 1 μmol of the conjugate of GSH with CDNB per min. The total protein concentration in the homogenate was measured using the method of Bradford.

HepG2 cells at the log phase were prepared as single-cell suspensions and seeded into 6-well plates (1 × 10⁶ cells/well) at 37°C for 24 h. After treatment with the EAF for 24 h, the cells were incubated with H₂O₂ for 4 h, washed two times with a PBS solution and lysed in lysis buffer (Biyuntian Biotechnology Co., Ltd, Shanghai, China). A BCA protein assay kit (Jiancheng Bioengineering Institute, Nanjing, China) was used to measure the intracellular protein content. The malondialdehyde (MDA) content, total antioxidant ability (T-AOC), catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), Na⁺/K⁺-adenosine triphosphate (Na⁺/K⁺-ATP) and Ca²⁺/Mg²⁺-adenosine triphosphate (Ca²⁺/Mg²⁺-ATP) enzyme activity were determined with the corresponding assay kits according to the manufacturer's instructions (Jiancheng Bioengineering Institute, Nanjing, China).