Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates.

In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.

Efeito in vitro de inibidores de proteases sobre trofozoítos de Giardia duodenalis / In vitro effect of protease inhibotors on trophozoites of giardia duodenalis

Entre os alvos mais promissores para o desenvolvimento de novos agentes antiparasitários, destacam-se as proteases que nos protozoários participam de processos metabólicos e fisiológicos, atuando como importantes fatores de virulência. Como a atividade dessas moléculas pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial terapêutico em diferentes infecções parasitárias, inclusive por Giardia. O presente estudo foi desenvolvido com o objetivo de avaliar o efeito in vitro dos inibidores de cisteína (IAA e E-64) e serina-proteases (antipaina, leupeptina e TLCK) sobre o crescimento, aderência, viabilidade e ultraestrutura de trofozoítos de cepa de Giardia isolada e axenizada em Botucatu. Para isso, trofozoítos foram incubados em meio contendo os inibidores a diferentes concentrações durante 24, 48 e 72 horas. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado a partir de contagens em hemocitômetro, enquanto que a viabilidade celular e as alterações ultraestruturais foram avaliadas, respectivamente, pelo método de redução do MTT e por microscopia eletrônica de transmissão. De acordo com as observações feitas no presente estudo, todos os inibidores de proteases apresentaram efeito sobre o crescimento, aderência e viabilidade dos trofozoítos. Entretanto, melhor desempenho quanto à capacidade de reduzir os parâmetros avaliados foi demonstrado nos ensaios com os inibidores de cisteína-proteases, especialmente a IAA. As maiores porcentagens de inibição do crescimento e aderência e as menores taxas de viabilidade foram observadas após o tratamento com IAA...

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In light of this, the proteolytic enzymes or proteases have excited the researcher’s interest, once they have been identified as important virulence factors as well as potential chemotherapeutic targets in parasites. Considering that proteases are naturally regulated by specific inhibitors, these substances have been evaluated for their therapeutic potential in parasitic infections including Giardia. In this way, we proposed to evaluate the in vitro effect of inhibitors of cysteine (IAA and E-64) and serine proteases (antipain, leupeptin and TLCK) on growth, adherence, viability and ultrastructure of Giardia trophozoites of a strain isolated and axenized in Botucatu. For this, trophozoites were incubated in medium containing the inhibitors at various concentrations for 24, 48 and 72 hours. In growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability and ultrastructural changes were evaluated, respectively, by the method of MTT and transmission electron microscopy. In this study, all protease inhibitors showed effect on growth, adherence and viability of trophozoites. However, better performance in their ability to reduce the parameters assessed was demonstrated in experiments with cysteine proteases inhibitors, especially IAA...

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae).

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ pump by a lipid probe that senses membrane domain changes.

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

Properties of a Kunitz-type trypsin inhibitor from Delonix regia seeds against digestive proteinases of Anagasta kuehniella (Z.) and Corcyra cephalonica (S.) (Lepidoptera: Pyralidae).

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

Morphology and preliminary enzyme characterization of the salivary glands from the predatory bug Podisus nigrispinus (Heteroptera: Pentatomidae).

Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom.

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 microM/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development.

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.

In vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611.

Hemolytic activity was evaluated in the putative periodontopathogens Prevotella intermedia and Prevotella nigrescens. Whole cells of both species present weak hemolytic activity evidenced only by solid media assays after 48 h of bacterial growth or after 5 h of interaction with erythrocytes at 37 degrees C in liquid assays. In this work we show that the use of crude extract allowed the detection of a higher hemolytic activity for P. intermedia, but surprisingly not for P. nigrescens. Incubation at 37 degrees C for 9 h, or treatment with trypsin or proteinase K, increased or exposed the hemolytic activity of P. intermedia and P. nigrescens crude extract, respectively. The activation process was inhibited by TLCK and PMSF but not by EDTA, E-64 or pepstatin A, indicating the serino-protease nature of the factor involved in activation of P. intermedia and P. nigrescens hemolysins. Both the buffer and the pH employed for cell fractionation influenced the activation of hemolysin, and the best results were obtained with Universal buffer at pH 8.0. The activated hemolysins acted optimally at pH 6.5 at 37 degrees C and the maximum hemolytic activity was detected at the early log phase of growth. The results of this study show for the first time a strong hemolytic activity for P. nigrescens and evidence of proteolytic activation of hemolysins produced by periodontopathogens.

The 26S proteasome participates in the sequential inhibition of estrous behavior induced by progesterone in rats.

Estrous behavior induced by progesterone (P) treatment of estradiol-primed rats is followed by a period in which females do not respond behaviorally to a second administration of P [sequential inhibition (SI)]. SI is thought to involve P-dependent down-regulation of hypothalamic P receptor (PR) content. This study tested the hypothesis that the 26S proteasome participates in the regulation of SI and brain PR content in female rats. Ovariectomized, estrogen-primed (estradiol benzoate, 2 microg s.c.) adult rats were injected with P (1 mg s.c.) alone or P with the proteasome inhibitors Z-Ile-Glu (OBu(1))-Ala-Leu-H (PSI, 300 microg/100 g s.c.) or N alpha-tosyl-lysyl chloromethyl ketone (TLCK, 200 microg i.p.) administered 48 h after estradiol priming. Sexual behavior was assessed in all animals 4 h later. These two agents inhibit 26S proteasome-mediated protein degradation by different mechanisms. To explore SI, the animals received a second P injection 24 h after the first, and a second sexual behavior test was performed 4 h later. After this test, brains were excised, and proteins were extracted from the preoptic area and the hypothalamus and processed for semiquantitative immunoblotting. In the first sexual behavior test (facilitation test), all animals treated with estradiol + P exhibited intense lordosis behavior. In the second sexual behavior test (inhibition test), both lordosis and proceptivity were significantly reduced in response to the second administration of P (SI). The magnitude of SI was significantly attenuated by the administration of either PSI or TLCK concurrently with the first P injection. The first P injection reduced PR content in the hypothalamus but not in the preoptic area. In contrast, PSI and TLCK significantly increased PR content in both structures. Our results suggest that PR degradation by the 26S proteasome participates in the expression of P-induced SI in female rats.

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting.

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.

Purification and characterization of a fibrinogen-clotting enzyme from the venom of jararacuçu (Bothrops jararacussu).

A clotting enzyme of the venom of Bothrops jararacussu, denoted FC-Bj, was purified by gel chromatography on Sephadex G-100 followed by HPLC on DEAE-5PW-PAK and gel filtration on Sephacryl S-200HR. The enzyme was identified as an acidic glycoprotein which probably consists of a single polypeptide chain with isoelectric point values in the range 3.3-4.4 and containing approx. 19% carbohydrates. On polyacrylamide gel electrophoresis (PAGE) at pH 8.3, the enzyme presented a diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the enzyme showed two protein bands corresponding to mol. wts of 50,600 and 60,000. After treatment of the enzyme with neuraminidase, a strongly stained band and a band weaker in staining intensity were observed on SDS-PAGE, thereby reducing the mol. wts to 44,500 and 56,300, respectively. The clotting factor possessed N-alpha-benzoyl-DL-arginine p-nitroanilide hydrolysing activity and coagulated fibrinogen to fibrin. These activities were 0.548 units/mg and 50.55 NIH thrombin units/mg, respectively. The proteinase was of the serine type, as indicated by sensitivity to phenylmethanesulfonyl fluoride and benzamidine. However, the amidolytic activity of this enzyme was resistant to inhibitors such as heparin, aprotinin, agmatine, EDTA, I-2581 and TLCK. The importance of disulfide bridges for the structural integrity of the purified enzyme was indicated by the loss of amidolytic activity in the presence of beta-mercaptoethanol and dithiothreitol. SDS-PAGE of fibrinogen degraded with this enzyme revealed the disappearance of the A alpha and B beta chains and the appearance of lower mol. wt fragments. The enzyme was able to hydrolyse synthetic chromogenic substrates with arginine as the C-terminal residue, and the kinetic parameters were determined. It hydrolysed the plasma kallikrein substrate H-D-Pro-Phe-Arg-pNA (S-2302) and produced kinin-releasing activity causing ileum contraction. In addition, hypotension and bradycardia were observed in urethane-anesthetized rats upon i.v. injection of the enzyme.