640 presence of cysteine proteinases in the glands might be expected in some other species of the Strigeidae family. This is why the present histochemical and biochemical inves- tigations were undertaken. Materials and methods Histochemistry Cercariae of C. cornutus, which emerged from snails Planorbarius corneus were concentrated by sedimentation in tap water at 1°C. Small drops of a dense suspension of the larvae were then transferred onto microscope slides. Excess water was removed and the larvae were kept under a stream of air until dry. Following fixation in a mixture of acetone and chloroform (2:1 or 2:0.6 by volume) for 1–2 min at room temperature, the larvae were covered with a drop of a histochemical medium comprising 0.05 M acetate buffer or 3,3′-dimethylglutaric acid-NaOH buffer at pH 5.2–5.8, 1 mM 5-nitrosalicylaldehyde (initially dissolved in N,N- dimethylformamide), 0.5–1.5 mM dithioerythritol (DTE), 0.5 mM disodium ethylenediaminetetraacetate (EDTA), and 0.1 mM Z-Arg-NHnapMeO (Dolbeare and Vanderlaan 1979). Control specimens were pretreated for 10 min with 0.1 mM N-ethylmaleimide (NEM) in the buffer. Following 30–45 min of an incubation in the dark, in a moist chamber, the material was examined under a fluorescence microscope for the presence of insoluble fluorescing deposits resulting from coupling of the liberated 4-methoxy-2-naphthylamine to 5-nitrosalicylaldehyde. Preparation of an extract Approximately 100 mg (fresh weight) of pelleted larvae was suspended in 10 mM sodium chloride solution and disrupted by application of three 4-sec ultrasonic bursts at 100 W (Braun sonifier, model Labsonic L) with intervening cooling at 0°C. Following centrifugation (10 min, 16,000 × g, 4°C) the supernatant was used as a source of proteolytic enzymes. Protein concentration was measured with the Coomassie blue protein quantitation method according to Bradford (1976) using crystalline bovine serum albumin as standard. Assay of proteolytic activity Proteolytic activity in the extract was examined at pH 5.2–9.6 (at 0.4 pH intervals), using azoalbumin, azocasein, and azocoll as substrates (Tomarelli et al. 1949; Charney and Tomarelli 1947; Moczoń 1999). An elastinolytic activity toward elastin–orcein (Schotton 1970) was assayed at pH 6.2–9.6. 3,3′-Dimethylglutaric acid-NaOH buffer (pH 5.0–7.2), Tris– Parasitol Res (2011) 108:639–643 HCl buffer (pH 7.4–8.8), and CHES–NaOH buffer (pH 9.2– 9.6) covered the whole pH range. Assay mixtures, 300 μl of volume, comprised a 0.07-M buffer, 2 mg of a substrate, 1 mM EDTA, 1 mM DTE, and 13 μg of extract protein. Incubation time was 60 min (for azoalbumin, azocasein, and azocoll), and 24 h (for elastin–orcein), at 24°C. Control mixtures were supplemented with the extract at the end of the incubation time. Peptidase activity was measured spectrophotometrically at the pH range of 5.2–9.6. Incubation media comprised the above mentioned EDTA–DTE buffers, 55 μM N-benzoyl-L- arginine-4-nitroanilide (Bz-Arg-NHnan) as a substrate and 15.5 μg of extract protein. For control, DTE was replaced with 0.1 mM pHMB. Following 10 min of incubation at room temperature, optical density was read against an extract-free blank. A molar extinction coefficient ε410= 8,800 was used for calculation of numbers of nanomoles of the substrate hydrolyzed. Electrophoresis Native electrophoresis was performed at 4°C at an operative pH of 3.5 (Reisfeld et al. 1962) in a polyacryl- amide gel (linear gradient, T= 5%, C = 2% to T= 18%, C = 3.5%; notation of Hjertén 1962) containing 0.1% gelatin. For control, extract samples treated with 0.1 mM NEM for 10 min were run in parallel to the untreated ones. After separation, fragments of gel slabs were rinsed in distilled water and transferred to a 0.05-M Tris–maleate–NaOH buffer, pH 6.8 which was supplemented with 1 mM EDTA and 5 mM 2-mercaptoethanol. Following incubation for 90 min at 24°C under continuous but moderate shaking, the gels were routinely fixed, stained with Coomassie brilliant blue, and examined for transparent zones indicating proteolytic activity. Activation/inhibition Studies on activation/inhibition were performed with the use of azocoll at pH 7.2 as described above. Extract samples were preincubated for 10 min in the presence of potent proteinase activators/inhibitors prior to the addition of the substrate. A series of appropriate controls (extract was added after removal of the unhydrolyzed substrate) was processed in parallel to the assay mixtures. Results Histochemistry Golden-yellow fluorescing precipitates were observed in those areas of the body of the cercariae which were