Patients, material and methods 37 To study consequences of oxidative stress several fibroblast cultures of Family W were treated with the superoxide generator paraquat (0.5mM for 48h). 2.3.6 Mitochondrial preparation Mitochondria were isolated from fibroblasts as previously described by Almeida and Medina (1997). Cells were removed from flasks by incubating the cells with 0.5% (wt/vol) trypsin resuspended in versene for 5min. Trypsinisation was stopped by addition of an equal volume of isolation medium supplemented with 10% (vol/vol) foetal bovine serum. The cells were pelleted by centrifugation at 1000rpm for 5min. (The pellet can be frozen at -80°C at this point.) To proceed, cells were resuspended in 1ml ice-cold homogenization buffer. Cells were then centrifuged at 4000 x g for 5min and the supernatant was discarded. The pellet was again resuspended in 2ml ice-cold homogenisation buffer. Cells were optimally homogenised on ice by 20 strokes of a tight fitting glass-teflon homogeniser revolving at 1000rpm. Cell homogenates were centrifuged at 1500 x g for 10min at 4 ºC, the supernatant placed on ice, and the pellet resuspended in 2ml homogenisation buffer, homogenised and centrifuged as above. The supernatants were then combined and centrifuged once more at 1500 x g (10min, 4ºC). The pellet was discarded, and the supernatant centrifuged at 11500 x g for 12min at 4ºC. The received mitochondria pellet was resuspended in 75μl homogenization buffer, frozen in liquid nitrogen, and stored at –80ºC until required. The activity of the mitochondrial marker enzyme citrate synthase was enriched approximately 3-fold between the initial cell homogenate and the final mitochondrial pellet. 2.3.7 Protein determination Sample protein concentration was determined by use of the BCA protein assay kit according manufacture’s protocol. Absorbance was measured on a plate reader at 562nm. Sample protein concentration was calculated from the BCA standard calibration curve (0-1500μg/ml). 2.3.8 Mitochondrial enzyme assays The activities of complexes I to IV, of citrate synthase and of malate dehydrogenase were determined in mitochondrial preparations by means of a spectrophotometical approach. The data of the complexes were expressed against citrate synthase.
Patients, material and methods 38 220.127.116.11 Citrate synthase assay The enzyme citrate synthase exists in nearly all living cells and stands as a pace-making enzyme in the first step of the Citric Acid Cycle. Citrate synthase is localized within eukaryotic cells in the mitochondrial matrix, but is encoded by nuclear DNA rather than mitochondrial (Wiegand and Remington, 1986). It is therefore used as a cellular marker for mitochondrial integrity and content. Citrate synthase activity (CS) was determined using a Hitachi U-3310 spectrophotometer as described by Coore et al. (1971). Sample (10-20μg protein; freeze-thawed three times in liquid nitrogen) was mixed with 100mM Tris (pH 8.0), 0.1mM acetyl coenzyme A, 0.1% (wt/vol) Triton-X100, and 0.1mM DTNB in a cuvette (total volume 1ml, path length 1cm). The reaction was started by the addition of 0.1mM oxaloacetate, and activity measured at 412nm for 5min at 30ºC (DTNB extinction coefficient = 13.6 x 10 3 M -1 cm -1 ). Samples were run against a reference cuvette that contained sample and all substrates except oxaloacetate. Citrate synthase activity was linear between 5 and 25μg protein. 18.104.22.168 Malate dehydrogenase assay Like citrate synthase, malate dehydrogenase is an enzyme of the Citric Acid Cycle where it catalyzes the conversion of malate into oxaloacetate and vice versa. In eukaryotes two different isoforms of the enzyme can be found – a mitochondrial and a cytoplasmic version (Davidson and Cortner, 1967). The activity of the mitochondrial malate dehydrogenase (MDHM) is used to determine the mitochondrial content in cells. MDHM was measured according to the method of Lai and Clark (1976). The reaction mixture contained 100mM potassium phosphate buffer, pH7.4, 0.16mM NADH, 0.16% (v/v) Triton X-100, 133pM oxaloacetate and about 10μl of the cellular homogenates (cp. 2.3.6). After controlling the flattening of the baseline for 1-2min the reaction was commenced with the addition of oxaloacetate and the NADH oxidation was measured at 340nm for 5min at 30°C (NADH extinction coefficient = 6.81 x 10 3 M -1 cm -1 ; total volume = 1ml; path length = 1cm). Homogenates were measured against a reference cuvette that contained all components except oxaloacetate. 22.214.171.124 Complex I assay (NADH dehydrogenase) Complex I activity (CI) was determined spectrophotometrically using a Hitachi U-3310 spectrophotometer as described by Ragan et al. (1987). Sample (10-20μg protein; freezethawed three times in liquid nitrogen) was mixed with 20mM phosphate buffer (pH 7.2), 2.5mg/ml BSA, 0.15mM NADH, and 1mM KCN in a cuvette. The reaction was started by the