Marc H. Dresden

Schistosoma mansoni: Proteinase activity of “hemoglobinase” from the digestive tract of adult worms

A method of collecting samples from the Schistosoma mansoni digestive tract was used to study proteinase activity. Activity against hemoglobin and a low molecular weight synthetic substrate, carbobenzoxy-arginyl-arginyl-7-amino-4-trifluoromethylcoumarin, was demonstrated in the soluble fraction of material regurgitated by S. mansoni adults and was dependent on the addition of a thiol compound, cysteine, to the assays. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography (AcA54), two proteins with estimated mol wt of 32,500 and 28,500 were found in the regurgitant and were associated with proteinase activity against both hemoglobin and the synthetic substrate. Homogenates of intact worms showed greater specific activity (synthetic substrate) in the females. Further, in bisected worms proteinase activity paralleled protein content, suggesting that, once secreted into the lumen, proteinase activity was distributed throughout the worm digestive tract.

Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase".

Abstract This report presents evidence that the “hemoglobinase” from adult Schistosoma mansoni, first described by Timms and Bueding and later by Senft and his collaborators, belongs to the class of thiol proteinases. Proteolytic activity is stimulated by SH-containing compounds and inhibited by N-ethylmaleimide as well as other inhibitors of thiol proteinases. The enzyme can be partially purified by affinity chromatography using a Sepharose-linked organomercurial ligand. In addition to its activity on globin and hemoglobin, the enzyme can also be assayed with Azocoll, a general protease substrate, and by the activation of inactive trypsinogen to active trypsin. Extraction of the enzyme is enhanced by the addition of the nonionic detergent Triton X-100.

Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

Adult Fasciola hepatica worms contain multiple proteinases capable of degrading hemoglobin, immunoglobulins and collagen. Here we report the isolation and biochemical characterization of a cysteine proteinase from acidic extracts of these worms. The enzyme was purified to homogeneity by cation exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 14,500 and migrated as a single band at approximately 14,500 Da upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Activity was assessed by employing synthetic peptide substrates, such as carbobenzoxy-phenylalanyl-arginyl-7-amino-4-trifluoro-methylcoumarin, commonly used to assay other cysteine proteinases. The proteinase was maximally active at pH 6.0, with 50% or more of the activity detected between pH 4.5 and 7.5. Inhibition of activity at pH 5.5 was seen only with compounds known to inhibit cysteine proteinases. No effect was seen with inhibitors of aspartic, serine, or metalloproteinases. The purified enzyme was stable at acidic pH at 4 degrees C, 25 degrees C, -20 degrees C, and in 1 M urea.

Purification of cysteine proteinases from adult Schistosoma mansoni

Proteolytic activity against hemoglobin and low molecular weight synthetic substrates has been previously found in homogenates and excretion/secretion products of adult Schistosoma mansoni worms. This activity is stimulated in the presence of thiol compounds and is maximally active at acidic pH. To characterize further this proteolytic activity, lyophilized adult worms were extracted, and proteinases were isolated and purified. From extracts prepared in 0.2 M citrate buffer, pH 4.9, two proteinase species were purified to homogeneity by centrifugation, gel filtration, dialysis, and chromatofocusing chromatography. The proteinases, designated SMw32 and SMw28, have apparent molecular weights (SDS-PAGE) of 31,700 +/- 1400 and 27,800 +/- 1700, respectively. Both are thiol-dependent, acidic endopeptidases that cleave hemoglobin and a synthetic substrate, CBZ-arg-arg-AFC. A statistical comparison of amino acid compositions reveals that the proteinases are highly related.

Strongyloides ransomi: Proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: synthesis, accumulation, and maturation.

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis. Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [3H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.

Schistosoma mansoni: Effect of some cations on the proteolytic enzymes of cercariae

Abstract The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na + and K + was demonstrated, with maximum stimulation at 20–40 m M concentrations. The divalent cations Mg 2+ and Ca 2+ stimulated proteolytic activity at low concentrations (between 0 and 10 m M ) but inhibited activity at higher concentrations. The divalent cations Zn 2+ , Cu 2+ , Fe 2+ , and Co 2+ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.

Properties of the acid thiol proteinase from Schistosoma mansoni adults

Seven beta-naphthylamine-linked peptides were tested as substrates for a previously described thiol proteinase of adult Schistosoma mansoni. The enzyme was not active on carbobenoxy-arginyl-arginyl-4-methoxy-2-naphthylamide and carbobenzoxy-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide. Enzyme activity was maximal at acidic pH (4.9-5.5) with similar optima for both macromolecular and peptide substrates. Activity of partially purified enzyme preparations against carbobenzoxy-arginyl-arginyl-4-methoxy-2-naphthylamide was stimulated more than 10-fold by thiols. The properties of this proteinase differ from those of proteolytic enzymes from the cercariae ad eggs of S. mansoni.

Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms.

1. Adult Clonorchis sinensis, the Chinese liver fluke, is known to migrate to the bile ducts of its mammalian host and cause significant pathology. 2. An acidic, thiol-dependent proteinase with a native mol. wt of approximately 18,500 was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. By SDS-polyacrylamide gel electrophoresis, the mol. wt of the enzyme was estimated to be 15,000. 3. The enzyme was similar to cathepsin B-like cysteine proteinases based on pH optimum, substrate specificity, and inhibitor sensitivity. 4. Antisera from human clonorchiasis and C. sinensis-infected rabbits reacted in immunoblots with the partially purified proteinase. The C. sinensis proteinase may be useful for serodiagnosis of clonorchiasis.

Schistosoma mansoni: Calcium Content of Cercariae and Its Effects on Protease Activity In vitro

The localization and quantitation of calcium in the protease-containing preacetabular glands of Schistosoma mansoni cercariae were determined by electron probe analysis and atomic absorption spectroscopy. Levels of calcium in these glands appear to exceed 8 to 10 M. In vitro, these high levels of calcium inhibit the cercarial proteases reversibly. It is suggested that calcium functions to control protease activity in situ in these organisms. The infective stage of the human blood fluke, Schistosoma mansoni, infects its host by penetrating the skin. The penetration process is thought to be mediated by proteolytic enzymes present in the preacetabular glands of this organism (Lewert and Lee, 1956; Stirewalt and Fregeau, 1966; Gazzinelli et al., 1966; Dresden and Asch, 1972). Moreover, it has been demonstrated that these glands contain large amounts of calcium (Stirewalt, 1959; Lewert et al., 1966). Lewert and Lee (1956) originally reported that these proteases are stimulated at low concentrations of these cations and we have confirmed this observation (Dresden and Edlin, 1974). Recently we determined that the proteolytic enzymes in extracts of this organism are inhibited by high concentrations of calcium and magnesium (Dresden and Edlin, 1974). We suggested that calcium might play a significant role in controlling the activity of these proteases by acting as an inhibitor of protease activity in situ. The results described in this report provide qualitative and quantitative evidence on the levels of calcium in these glands and its effects on protease activity. MATERIALS AND METHODS Infected snails, Biomphalaria glabrata (Puerto Rican strain), were allowed to shed their cercariae at room temperature in beakers containing dechlorinated tap water or 1 mM Tris-HCl, pH 7.8. Using a depression slide and a dissecting microscope, individual cercariae were transferred by disposable pipette. For electron probe analysis single cercariae were dried on silica discs at room temperature as rapidly as possible, aided by withdrawal of water with a micropipette. For calcium analysis, individual cercariae (50 Received for publication 10 October 1974. t 200) were counted and transferred to appropriate tubes. Spectrophotometric analysis of calcium was by the method of Williams and Wilson (1961). CaCl2 and CaCO3 (0.5 to 100 ,ug) were used as standards. Atomic absorption spectroscopy for calcium was determined following extraction of 100 to 200 cercariae in 5% lanthanum chloride-1% HC1 at 100 C for 10 min. Calcium contributed by sources other than the cercariae was corrected for by using uninfected snails. Protease activity in cercarial extracts was measured using Azocoll substrate as described previously (Dresden and Edlin, 1974). RESULTS AND DISCUSSION The results of the electron probe X-ray analysis and scanning electron microscopy are shown in Figure la-c. These figures represent a single cercaria examined by scanning electron microscopy (Fig. la) and by X-ray probe analysis for calcium (Fig. lb) and phosphorus (Fig. lc). A number of individual cercariae were examined by these combined techniques. The results demonstrate dramatically a concentration of calcium in the region of the preacetabular glands, and a fairly uniform distribution of phosphorus throughout the body of the cercariae. Using the tail region as a control, scanning X-ray analysis was carried out for 20to 40-sec intervals at 25 kv with a 0.185-,uamp sample current. These experiments demonstrated that calcium is at least 1,000 to 2,000 times more abundant in the preacetabular gland region than in the tail region. Magnesium levels in the gland region were not significantly elevated over those in the tail region. While these experiments established the localization of calcium, it would have been necessary to determine the thickness and composition of the cercariae in order to determine