Fish
Two-year-old brown trout males (n = 6) were collected by random net fishing from aquaculture pools (Posto Aquícola do Torno – Amarante, Portugal) in late February. Fish of 188 g (coefficient of variation, CV = 0.10) in weight and 26 cm (CV = 0.04) in length were kept in dechlorinated water (pH 6–7, at 8–10°C) for 1–2 days before sacrifice. Mean liver weight was 3.0 g (CV = 0.30).
Chemicals
Cofactors, substrates for enzyme assays and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (Poole, Dorset, U.K). Other chemicals were obtained from Merck (Darmstadt, Germany).
Preparative procedures
Animals were anaesthetised by bathing in a solution of ethylene glycol monophenyl ether (0.4 ml l-1) and then perfused at 5.6 ml min-1 kg-1 via the portal venous system for 4–5 min with an isosmotic buffer for salmonids [20]. The liver was weighted just after removal, immediately minced in chilled homogenization buffer with a pH of 7.4 (250 mM sucrose, 5 mM MOPS, 1 mM EDTA and 0.1% ethanol saturated with phenyl-methyl-sulfonyl-fluoride PMSF) [17] and then homogenized in the same buffer using a Potter-Elvehjem homogenizer at 1000 rpm, held in an ice-water-bath. After sedimentation of unhomogenized material (50 g, 10 min, 4°C) according to Cajaraville et al. [21], supernatant volume was adjusted to 5 ml g-1 of liver using ice-cold homogenisation buffer (A fraction). A previously described method [17] was used for differential pelleting of the A fraction in order to produce a B fraction containing mainly mitochondria and lysosomes (2,000 g, 10 min, 4°C), a D fraction enriched with peroxisomes (20,000 g, 30 min, 4°C), a E fraction enriched with microsomes (100,000 g, 60 min, 4°C) and a cytosolic F fraction (supernatant of 100,000 g, 60 min, 4°C). Pellets (B, D and E) were rinsed once by resuspension in an appropriate volume of buffer using a glass-rod and recentrifuged under the same conditions. Before enzymatic assay, all fractions were treated with Triton X-100 at a final concentration of 0.5% (v/v) in homogenization buffer. Proteins were assayed according to Lowry et al. [22] using BSA standards and results are expressed in BSA equivalents (Table 1).
Enzyme assays
Enzymatic assays were carried out in a spectrophotometer connected to a circulating water system for temperature regulation in the cuvette compartment. Enzyme activities linear in time and proportional to the amount protein in the assays were obtained using an appropriate sample dilution. Two different dilutions of each sample were used to produce a mean value of enzymatic activities. Temperature effect on peroxisomal enzyme activities was evaluated by measurements made at 10, 15, 20, 25, 30 and 37°C.
Catalase
For catalase the methodology previously described by Aebi [14] was used. Incubation medium contained 50 mM sodium phosphate buffer (pH 7), 10 mM hydrogen peroxide (H2O2) and a diluted sample. Consumption of H2O2 was followed by the decrease in absorbance at 240 nm for 30 seconds and the activity is expressed by the first-order rate constant (k) for degradation of H2O2. k = (1 ÷ Δt) × ln (c1 ÷ c2) where c1 and c2 correspond to H2O2 concentrations at t = 0 and t = 30 seconds, respectively [14].
Peroxisomal oxidases
Measurement of peroxisomal oxidase activities was based on the production of H2O2 and followed the procedure of Cablé et al. [13] with some modifications. Incubation medium contained 50 mM potassium phosphate buffer (pH 8.3), 0.082 mM 4-amino-antipyrine, 1 mM phenol and 2 IU ml-1 of horseradish peroxidase. To avoid the interference of catalase, 10 mM of azide was added to the medium, according to Leupold et al. [15]. For glycolate oxidase assay, 0.01 mM flavin mononucleotide (FMN) was added in the medium [23]. For other oxidases, 0.01 mM flavin adenine dinucleotide (FAD) was included in the medium [13, 23]. Substrates were used in the following concentrations: 20 mM D-alanine, 1 mM uric acid, 0.1 mM palmitoyl-CoA (C:16) and 20 mM sodium glycolate. Each enzymatic reaction was started by addition of 25 μl of a diluted sample to 650 μl of incubation medium. Absorbance increase was measured at 500 nm for 10 min. For urate oxidase, a baseline was made with the complete medium without sample, which was thereafter subtracted from the absorbance increase in each assay. This procedure was not necessary for all other assayed oxidases, because non-specific reactions were not detected in other cases.
The amount of H2O2 (in μM) produced by peroxisomal oxidases was calculated from the equation of the calibration line: [H2O2] = 185.07 × Absorbance at 500 nm. This line (Fig. 3) showed a regression coefficient of 1.0 and was constructed using several standards of H2O2, which were added to the incubation medium without sample. The concentration of the H2O2 standards was calculated from the absorbance at 240 nm (ε = 39.4 M-1 cm-1) [24].
Succinate dehydrogenase
Succinate dehydrogenase activity was estimated according to Schoner et al. [25]. Incubation medium contained 50 mM potassium phosphate buffer (pH 7.5), 5 mM sodium succinate, 1 mM potassium cyanide, 0.1 mM 2,6-dichlorophenol indophenol (DPIP), and a diluted sample. The extinction coefficient of DPIP at 600 nm, determined with several standard solutions ranging from 0 to 100 μM of DPIP (ε = 17.3 mM-1 cm-1), was used to calculate the activity [25].
Aryl sulphatase
Measurements of aryl sulphatase activity followed the method of Worwood et al. [26]. Incubation medium contained 0.5 M acetate buffer (pH 5.6), 20 mM nitrocatechol sulphate and a diluted sample. The reaction was stopped by addition 1 ml of 1 M sodium hydroxide and the extinction coefficient of nitrocatechol solutions at 515 nm (ε = 12.4 mM-1 cm-1) was used to calculate the activity [26].
NADPH cytochrome c reductase
Measurements of NADPH cytochrome c reductase activity followed the method of Johannesen et al. [27]. Incubation medium contained 0.1 M TRIS-HCl buffer (pH 7.6), 10 mM potassium cyanide, 0.4 mM oxidised cytochrome c, 3 mM NADPH and a diluted sample. This reaction was started by addition of NADPH. Activity calculations were based upon the extinction coefficient of a reduced cytochrome c solution at 550 nm (ε = 19.6 mM-1 cm-1) [28].
Calculations, units and statistical analysis
The percentage distribution [(protein or enzyme activity in one fraction ÷ Σ of protein or enzyme activity in fractions B to F) × 100] and the relative specific activity [percentage distribution of the enzyme in one fraction ÷ percentage distribution of protein in that fraction] were calculated as in other papers [5]. Catalase activity is expressed by the first-order rate constant in s-1 g-1 of liver and s-1 mg-1 of protein, for all others, results are given in nmol min-1 g-1 of liver and nmol min-1 mg-1 of protein.
Data are reported as means per group of animals, followed by the respective coefficients of variations (CV = standard deviation ÷ mean). Correlation analysis was used to establish several relationships considered biologically or technically relevant. Results were judged significant when p ≤ 0.05 as reported by Pearson's coefficient of correlation (r). A linear regression analysis was made to study enzyme activities at six different chosen temperatures.