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A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).

Moczoń T - Parasitol. Res. (2010)

Bottom Line: The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin.The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it.The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure.

View Article: PubMed Central - PubMed

Affiliation: W Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland. moczon@twarda.pan.pl

ABSTRACT
A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.

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Gelatinolysis at pH 6.8 produced by the proteinases separated by electrophoresis at the operative pH of 3.5 at which the enzymes were practically inactive during migration
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Fig4: Gelatinolysis at pH 6.8 produced by the proteinases separated by electrophoresis at the operative pH of 3.5 at which the enzymes were practically inactive during migration

Mentions: 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 nmol min−1 μg−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 separation 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 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.Fig. 2


A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).

Moczoń T - Parasitol. Res. (2010)

Gelatinolysis at pH 6.8 produced by the proteinases separated by electrophoresis at the operative pH of 3.5 at which the enzymes were practically inactive during migration
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040816&req=5

Fig4: Gelatinolysis at pH 6.8 produced by the proteinases separated by electrophoresis at the operative pH of 3.5 at which the enzymes were practically inactive during migration
Mentions: 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 nmol min−1 μg−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 separation 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 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.Fig. 2

Bottom Line: The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin.The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it.The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure.

View Article: PubMed Central - PubMed

Affiliation: W Stefański Institute of Parasitology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland. moczon@twarda.pan.pl

ABSTRACT
A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.

Show MeSH
Related in: MedlinePlus