Molybdenum

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.

E. histolytica transformants expressing epitope-tagged mitosomal proteins were previously established [5] (link). Approximately 5×10⁵ trophozoites were resuspended in 2 ml BI-S-33 medium and seeded onto a molybdenum disk (Nissin EM Co., JAPAN) in a well of a 24-well plate. After 15-min incubation at 35.5°C, the molybdenum disk that amoebas adhered to was removed and immediately immersed in liquid propane at −175°C. The disk was further fixed and sectioned as previously described [21] (link). The disk was reacted with primary antibody diluted at 1∶2000 (anti-Cpn60 antiserum) and 1∶500 (anti-HA monoclonal antibody) in phosphate-buffered saline containing 1.5% bovine serum albumin for overnight at 4°C. The samples were then reacted with colloidal gold-conjugated anti-rabbit or anti-mouse secondary antibody (1∶20) for 1 h at room temperature. Samples were examined by electron microscopy at Tokaii Microscopy., Inc (Nagoya, JAPAN).