Parasitol Res (2011) 108:639–643 occupied by the penetration glands and by the secretory canals, both being filled with the secretion produced by the glandular cells (Fig. 1). The intensity of the reaction increased with the elevation of the pH, becoming optimal at the pH 5.6, the highest value at which the reaction product remains microcrystalline for a number of minutes and satisfactorily reflects the examined structures; however, from an unknown reason only one in approximately 100 of the examined specimens revealed a satisfactory pattern of the reaction. Due to the fixation and defatting, as well as to a low magnifying power, the activity of cathepsin B in the whole larval body was poorly perceptible. Enzyme activity The examination of proteolytic activity versus increasing pH values revealed two peaks, both being especially distinct in the case of the use of azocoll as the substrate (Fig. 2). The minor peak indicated the activity of cathepsin B. The major peak apparently illustrated the highest activity of the glandular proteinase at the pH range between 6.8 and 7.2. The glandular enzyme degraded azocoll, azocasein, and azoalbumin at the pH range of 5.2–8.8. Maximum rates of hydrolysis expressed as micrograms of the substrate per hour per microgram of extract protein, at an optimum pH, were 10.4 at pH between 6.8 and 7.2 for azocoll, 0.64 at pH 7.2 for azoalbumin, and 0.23 at pH 7.2 for azocasein. No hydrolysis of elastin–orcein was detected after 24 h of incubation at the pH range of 6.2–9.6. Bz-Arg-NHnan was split at a somewhat lower rate (8.6 nmolmin−1μg−1) at an optimum pH between 6.4 and 6.8 (Fig. 3). No liberation of p-nitroaniline was recorded in incubation mixtures in which DTE was replaced with pHMB. The electrophoretic separa- tion of extract proteins from the cercariae at the operative pH of 3.5, at which the proteinases were practically inactive, revealed the presence of two distinct transparent bands in the gel resulting from a relatively high gelatinolytic activity and three bands resulting from a weak activity (Fig. 4). The 641 Fig. 2 Rates of hydrolysis of protein substrates observed at differing pH in the presence of 1 mM EDTA and 1 mM DTE. Proteinase activity is expressed as microgram substrate per hour per extract protein. The left ordinate shows values obtained for azocoll (black circles). The right ordinate shows values obtained for azoalbumin (black triangles) and azocasein (empty squares). Each data point is a mean of duplicate determinations distinct bands apparently resulted from the activity of the glandular enzyme and cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis appeared in the control gel, thus indicating an irreversible modification of −SH groups of the enzymes by NEM. Activation/inhibition Calcium ions at 1 mM concentration reduced the proteinase activity by 12%, whereas 0.1 mM zinc sulfate inhibited it (Table 1). 1,10-Phenanthroline, a weak complexane of calcium and magnesium but a strong complexane of zinc, was without an inhibitory effect on the enzyme activity indicating the absence of a zinc-dependent activity of metalloproteinases. EDTA, a strong complexane of divalent and trivalent metal ions produced an increase in the activity Fig. 1 Hydrolysis of Z-Arg-NHnapMeO by the cysteine proteinase occupying the penetration glands (pg) and the secretory canals (sc) of the cercariae examined with the fluorescence method vs. ventral sucker; t, tail Fig. 3 Rates of hydrolysis of Bz-Arg-NHnan observed at differing pH in the presence of 1 mM EDTA and 1 mM DTE. Proteinase activity is expressed as nanomoles of the substrate per minute per microgram of extract protein. Each data point is a mean of duplicate determinations