Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes.

To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.

A novel butyrylcholinesterase from serum of Leporinus macrocephalus, a Neotropical fish.

We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.

Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis.

Extracellular proteolytic activity was detected in a Leishmania ( L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. ( L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.

Exploring the subsite specificity of Schistosoma mansoni aspartyl hemoglobinase through comparative molecular modelling.

Blood flukes of the genus Schistosoma currently infect millions of people in tropical and subtropical countries. An enzyme playing a major role in hemoglobin (Hb) degradation by Schistosoma mansoni has been cloned and shown to be highly similar to the human cathepsin D aspartyl proteinase, although presenting a distinct substrate specificity from the latter. Investigating the structural features responsible for this difference has a major application in the design of selective anti-schistosomal drugs. In order to achieve this goal a homology model for the S. mansoni aspartyl hemoglobinase was constructed and then used to simulate the complexes formed with two transition state analogues of Hb-derived octapeptide substrates. Comparison with human cathepsin D showed that different pocket volumes and surface electrostatic potentials arise from substitutions in residues comprising the S4, S3, S2 and S3' subsites. Since the primary specificity of the S. mansoni enzyme resembles that of HIV-1 protease, we have discussed the applicability of current retroviral protease inhibitors as leads for the design of new anti-schistosomal drugs.

Crithidia guilhermei: purification and partial characterization of a 62-kDa extracellular metalloproteinase.

An extracellular metalloproteinase from Crithidia guilhermei, a monoxenic trypanosomatid of insects, was purified 11-fold by ammonium sulfate precipitation, gel filtration on a Shinpack Diol-150 column, and anion-exchange chromatography in a MONO Q column, both using the HPLC system. The proteinase appeared as a single band with an apparent molecular mass of 62 kDa in SDS-PAGE, under reducing conditions, and was optimally active at 37 degrees C and pH 6.0. The enzyme showed 62% residual activity at 50 degrees C for 30 min. The proteinase was completely inhibited by 1, 10-phenanthroline, indicating that the enzyme belongs to the metalloproteinase class. This is the first report of the purification of an extracellular metalloproteinase from the Crithidia species. The possible role of this enzyme in the digestive tract of the insect host is discussed.

Detection of cysteine-proteinases in Leishmania amazonensis promastigotes using a cross-reactive antiserum.

Rabbit serum against the cysteine-proteinases papain has been employed for the cellular localization of cysteine-proteinases of in Leishmania amazonensis promastigotes. By immunocytochemistry, immune complexes were found in the plasma membrane and in the flagella pocket of the parasite. The antiserum immunoprecipitated major iodinated proteins with molecular masses of 66, 45, 28 and 24 kDa and a wide partitioning of the Triton X-114 detergent phase. The presence of cysteine-proteinase at the cell surface membrane was also suggested by the detection of proteolytic activity in living cells (19.0 microg azocasein min(-1) 10(-7) promastigotes (1.0 S.D. )).

Purification and partial characterization of creatine kinase from electric organ of Electrophorus electricus (L.).

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the electrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish (Torpedo) and human muscle type CK.

Purification and partial characterization of an extracellular serine-proteinase of Streptomyces cyaneus isolated from Brazilian cerrado soil.

Streptomyces cyaneus, a micro-organism isolated from Brazilian cerrado soil, produces an extracellular proteinase (SCP), which was purified 22-fold to homogeneity from culture supernatant fluid, using a single aprotinin-agarose affinity chromatography step. It is produced at a level corresponding to approximately 15% of total protein, but its physiological function has yet to be determined. The molecular mass of this S. cyaneus proteinase was estimated to be 120 kDa by gel filtration high performance liquid chromatography, and it migrates by SDS-PAGE as a single band of 30 kDa. It was optimally active at 25 degrees C and pH 9.0, and was fully inhibited by the serine-proteinase inhibitors PMSF and TPCK. A Km value of 1. 86 x 10-5 mmol l-1, and Vmax of 2.0 x 10-2 mmol l-1 (Abs247 nm microg-1 min-1), were calculated for alpha-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate.

Purification and partial characterization of glyceraldehyde-phosphate dehydrogenase from electric organ of Electrophorus electricus (L.).

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chromatography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectrophocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.

Purification and partial characterization of Trypanosoma cruzi triosephosphate isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on plenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isolectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytichemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins.

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster).

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0-6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-alpha-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-Bz-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-alpha and B-beta chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.

Detrimental effect of nitric oxide on Trypanosoma cruzi and Leishmania major like cells.

The production of nitric oxide (NO) by activated macrophages has been reported to be a non-specific immune-effect mechanism against several parasites. In this work we investigate whether the NO has a detrimental effect on the intracellular parasites of the genus Leishmania and as well as Trypanosoma cruzi. This was assessed by co-cultivating infective Leishmania promastigotes and T. cruzi trypomastigotes and non-infective T. cruzi epimastigotes forms of the parasites in the presence of the NO releasing molecule, S-nitroso-N-acetyl-DL-penicillamine (SNAP). We demonstrate that the NO has the ability to inhibits the growth of all parasites in a concentration dependent manner. In addition, by analysing purified protein and cell homogenates of L. major (promastigotes) and T. cruzi (epimastigotes and trypomastigotes) we demonstrated that the NO may regulate the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of both parasites.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing.

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDA protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Purification and partial characterization of a thrombin-like/gyroxin enzyme from bushmaster (Lachesis muta rhombeata) venom.

The acidic coagulating enzyme of the L. m. rhombeata venom was purified to homogeneity using one step on preparative isoelectric focusing followed by gel permeation on a high performance liquid chromatography system. The enzyme focused with pIs 3.1-5.0 and had a molecular mass of 47,000 mol. wt as determined by high performance liquid gel-filtration chromatography and about 45,000 mol. wt as judged by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The enzyme is a glycoprotein containing sialic acid and 12.4% of neutral carbohydrates. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein shows 100% identity with L. m. muta gyroxin and considerable sequence homology with gyroxin and thrombin-related proteins. The enzyme exhibits strong N-p-tosyl-L-arginine methyl esterase activity, hydrolyses tripeptide nitroanilide derivatives weakly or not at all, and cleaves specifically the fibropeptide A (alpha-chain).

Can software be used to predict antigenic regions in Plasmodium falciparum peptides?

We compared the antigenicity of p126 Plasmodium falciparum peptides with predicted antigenic regions identified using the methods described by Garnier et al. (1978) and Chou & Fasman (1974). For this purpose nine different P. falciparum peptides were synthesized in accordance with the deduced amino acid sequence of the p126 gene, and their reactivity was tested using an enzyme linked immunosorbent assay against sera from individuals with a natural malaria infection. Both predictive methods gave similar antigenic-index scores, however, a comparison of these predictive results with data obtained by ELISA showed that the probability of a correct prediction was only around 45% for both cases. Thus, in our view computer software could not be used in isolation for screening purposes, and other parameters must also be taken into account when using such software to assess antigenicity.

Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h.

Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h

A glass wool-based method for purifying Trypanosoma cruzi trypomastigotes and identification of an epimastigote-specific glass-adherent surface peptide.

Glass wool, hydrophilic cotton wool, non-electrically charged BIO-GEL P2 and common tissue paper columns were used to purify trypomastigotes from a mixed Trypanosoma cruzi population grown in axenic culture medium. With all these columns, highly purified (up to 98%) trypomastigote preparations were obtained. Trypomastigote yields from cotton wool, BIO-GEL P2 and common tissue paper columns were not as high as from glass wool columns, from which yields varied from 69 to 80%. Purification on glass wool did not affect trypomastigote infectivity or virulence. Dead trypomastigotes could not be purified on glass wool columns. A glass-adherent amphiphilic peptide of 45 kDa, present in the cell membrane, was isolated from epimastigote but not from trypomastigote preparations.

Isolation and immunological analysis of Trypanosoma cruzi glycolipids.

A water-soluble glycolipidic fraction from Trypanosoma cruzi was isolated using a mixture of hexane and isopropanol. Analysis by SDS-polyacrylamide gel electrophoresis, after staining by silver and Sudan Black B, showed that the fraction contained one band with a relatively high mobility. Its reactivity and specificity with human chagasic sera and T. cruzi infected mouse sera or with sera from patients with several other pathologies was determined by an immunoradiometric assay. The glycolipid-based radioimmunoassay for the detection of T. cruzi antigens provided a sensitive measure of its activity. However, cross-reactivity with several sera from patients with visceral and cutaneous leishmaniasis was detected.