The Kunitz chymotrypsin inhibitor from Erythrina velutina seeds displays activity against HeLa cells through arrest in cell cycle.

Erythrina velutina is a species of arboreal leguminous that occurs spontaneously in the northeastern states of Brazil. Leguminous seeds represent an abundant source of peptidase inhibitors, which play an important role in controlling peptidases involved in essential biological processes. The aim of this study was to purify and characterize a novel Kunitz-type peptidase inhibitor from Erythrina velutina seeds and evaluate its anti-proliferative effects against cancer cell lines. The Kunitz-type chymotrypsin inhibitor was purified from Erythrina velutina seeds (EvCI) by ammonium sulphate fractionation, trypsin- and chymotrypsin-sepharose affinity chromatographies and Resource Q anion-exchange column. The purified EvCI has a molecular mass of 18 kDa with homology to a Kunitz-type inhibitor. Inhibition assays revealed that EvCI is a competitive inhibitor of chymotrypsin (with K i of 4 × 10-8 M), with weak inhibitory activity against human elastase and without inhibition against trypsin, elastase, bromelain or papain. In addition, the inhibitory activity of EvCI was stable over a wide range of pH and temperature. Disulfide bridges are involved in stabilization of the reactive site in EvCI, since the reduction of disulfide bridges with DTT 100 mM abolished ~ 50% of its inhibitory activity. The inhibitor exhibited selective anti-proliferative properties against HeLa cells. The incubation of EvCI with HeLa cells triggered arrest in the cell cycle, suggesting that apoptosis is the mechanism of death induced by the inhibitor. EvCI constitutes an interesting anti-carcinogenic candidate for conventional cervical cancer treatments employed currently. The EvCI cytostatic effect on Hela cells indicates a promised compound to be used as anti-carcinogenic complement for conventional cervical treatments employed currently.

Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.

Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L. seeds with double activity toward serine- and cysteine-proteinases.

Kunitz proteinase inhibitors are widely distributed in legume seeds, and some of them have the ability to inhibit two different classes of enzymes. In this report, novel insights into three-dimensional structure and action mechanism of ApKTI, an Adenanthera pavonina Kunitz trypsin inhibitor, were provided to shed some light on an unconventional non-competitive activity against trypsin and papain. Firstly, ApKTI was purified by two tandem-size molecular exclusion chromatography high resolutions, Sephacryl S-100 and Superose 12 10/300 GL. Purified ApTKI showed molecular mass of 22 kDa and higher affinity against trypsin in comparison to papain, while the bifunctional inhibitor presented lower inhibitory activity. Moreover, in vitro assays showed that ApKTI has two independent interaction sites, permitting simultaneous inhibition to both enzymes. Theoretical three-dimensional structures of ApTKI complexed to both target proteinases were constructed in order to determine interaction mode by using Modeller v9.6. Since the structure of no non-competitive Kunitz inhibitor has been elucidated, ApTKI-trypsin and ApTKI-papain docking were carried out using Hex v5.1. In silico experiments showed that the opposite inhibitor loop interacts with adjacent sites of trypsin (Arg(64), Ser(107), Arg(88) and Lys(108)) and papain (Gln(51), Asp(172) and Arg(173)), probably forming a ternary complex. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. The predicted non-coincidence of trypsin and papain binding sites is completely different from that of previously proposed inhibitors, adding more information about mechanisms of non-competitive plant proteinase inhibitors.

Effects of a novel pathogenesis-related class 10 (PR-10) protein from Crotalaria pallida Roots with papain inhibitory activity against root-knot nematode Meloidogyne incognita.

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M(r) of 15 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K(i) value of 1.8 x 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC)-CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.

A lactose specific lectin from the sponge Cinachyrella apion: purification, characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes.

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca2+, Mg2+ and Mn2+ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 degrees C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.

A novel antimicrobial peptide from Crotalaria pallida seeds with activity against human and phytopathogens.

An actual severe problem in agriculture consists of an expressive increase of economical losses caused by fungi and resistant bacteria toward antibiotics. In order to find a solution to this problem, several studies have been concentrating on the screening of novel plant defense peptides with antimicrobial activities. These peptides are commonly characterized by having low molecular masses and cationic charges. The present work reports the purification and characterization of a novel plant peptide with molecular mass of 5340 Da, named Cp-AMP, from seeds of C. pallida, a typical plant from Caatinga biome. Purification was achieved using a size exclusion S-200 column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Cp-AMP was able to inhibit the development of filamentous fungi Fusarium oxysporum as well as the gram-negative bacterium Proteus sp. The identification of Cp-AMP could contribute, in the near future, to the development of biotechnological products, such as transgenic plants with enhanced resistance to pathogenic fungi and/or of antibiotics production derived from plant sources in order to control bacterial infections.

Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV).

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.

Effects of a chitin binding vicilin from Erythrina velutina seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus).

Erythrina velutina vicilin, EvV, is a dimeric glycoprotein with Mr of 124.6 kDa. EvV was tested for anti-insect activity against bean bruchid larvae. EvV had LD(50) of 0.10% and ED(50) of 0.14% for Z. subfasciatus and LD(50) of 0.26% and ED(50) of 0.19% for C. maculatus. EvV was not digested by bean larvae enzymes until 12 h of incubation, and at 24 h EvV was more resistant to Z. subfasciatus enzymes.

Larvicidal effects of a chitin-binding vicilin from Erythrina velutina seeds on the mediterranean fruit fly Ceratitis capitata.

Chitin-binding vicilin from Erythrina velutina seeds was purified by ammonium sulfate followed by affinity chromatography on a chitin column and gel filtration on Superose-6-10-300-GL. The Erythrina velutina vicilin, called EvV, is a tetrameric glycoprotein composed of 1.85% carbohydrates and M r of 216.6 kDa, consisting of two subunits of M r of 54.8 and two subunits of M r of 50.8 kDa. The EvV homogeneity was confirmed in native PAGE where it was observed to be a unique acid-protein band with slow mobility in this gel. Effect of EvV on C. capitata larvae was examined by bioassay and its mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV when added to diet caused strong effect on mortality (ED50 of 0.14%) and larval mass (WD50 of 0.12%). These deleterious effects were associated to the binding to chitin structures present in peritrophic membrane and to gut epithelial cells, and its low digestibility in C. capitata digestive tract. These results are the first demonstration of a proteinaceous bioinsecticide from plant origin effective against C. capitata larvae. EvV may be part of the pest management programs or an alternative in plant improvement program.

Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.

A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.

Effects of a chitin-binding vicilin from Enterolobium contortisiliquum seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus) and phytopathogenic fungi (Fusarium solani and Colletrichum lindemuntianum).

Chitin-binding vicilin from Enterolobium contortisiliquum seeds was purified by ammonium sulfate followed by gel filtration on Sephacryl 300-SH and on Sephacryl 200-SH. The vicilin, called EcV, is a dimeric glycoprotein composed of 1.03% carbohydrates and a Mr of 151 kDa, consisting of two subunits of Mr of 66.2 and 63.8 kDa. The EcV homogeneity was confirmed in a PAGE where it was observed to be a unique acid protein band with slow mobility in this native gel. E. contortisiliquum vicilin (EcV) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae and for phytopathogenic fungi, F. solani and C. lindemuntianum. EcV was very effective against both bruchids, producing 50% mortality for Z. subfasciatus at an LD50 of 0.43% and affected 50% of the larvae mass with an ED50 of 0.65%. In artificial diets given to C. maculatus, 50% of the larvae mass was affected with an ED50 of 1.03%, and larva mortality was 50% at LD50 of 1.11%. EcV was not digested by midgut homogenates of C. maculatus and Z. Subfasciatus until 12 h of incubation, and at 24 h EcV was more resistant to Z. subfasciatus larval proteases. The binding to chitin present in larvae gut associated to low EcV digestibility could explain its lethal effects. EcV also exerted an inhibitory effect on the germination of F. solani at concentrations of 10 and 20 microg mL-1. The effect of EcV on fungi is possibly due to binding to chitin-containing structures of the fungal cell wall.

Digestive enzymes during development of Ceratitis capitata (Diptera:Tephritidae) and effects of SBTI on its digestive serine proteinase targets.

The digestive system of Ceratitis capitata was characterized during its larval development and in the insect stage. Disaccharidases against maltose and sucrose were more evident in the 2nd and 3rd day of larval development and in the adult stage, respectively. Glycosil-hydrolyses with higher specific alpha-galactosidasic and beta-galactosidasic activities were detected in the 2nd and 3rd day of the larval stage, respectively. Specific proteolytic activities against azocasein showed an increase in the 4th and 5th day of the larval stage and in the adult stage. Specific hemoglobin activities were constant between 2nd and 6th day of the larval stage. The larvae used mainly serine proteinases, such as trypsin/chymotrypsin, and the adult insects only chymotrypsin-like enzymes in their digestive process. Two serine proteinases were separated from zymogram between the 4th and 5th day of larval development and in the adult stage. Effect of soybean trypsin inhibitor (SBTI, a serine proteinase inhibitor) on development of C. capitata was examined by bioassay. C. capitata was susceptible to SBTI which affected larval mass at ED50 3.01%.

In vivo bioinsecticidal activity toward Ceratitis capitata (fruit fly) and Callosobruchus maculatus (cowpea weevil) and in vitro bioinsecticidal activity toward different orders of insect pests of a trypsin inhibitor purified from tamarind tree (Tamarindus indica) seeds.

A proteinaceous inhibitor with high activity against trypsin-like serine proteinases was purified from seeds of the tamarind tree (Tamarindus indica) by gel filtration on Shephacryl S-200 followed by a reverse-phase HPLC Vidac C18 TP. The inhibitor, called the tamarind trypsin inhibitor (TTI), showed a Mr of 21.42 kDa by mass spectrometry analysis. TTI was a noncompetitive inhibitor with a Ki value of 1.7 x 10(-9) M. In vitro bioinsecticidal activity against insect digestive enzymes from different orders showed that TTI had remarkable activity against enzymes from coleopteran, Anthonomus grandis (29.6%), Zabrotes subfasciatus (51.6%), Callosobruchus maculatus (86.7%), Rhyzopertha dominica(88.2%), and lepidopteron, Plodia interpuncptella (26.7%), Alabama argillacea (53.8%), and Spodoptera frugiperda (75.5%). Also, digestive enzymes from Diptera, Ceratitis capitata (fruit fly), were inhibited (52.9%). In vivo bioinsecticidal assays toward C. capitata and C. maculatus larvae were developed. The concentration of TTI (w/w) in the artificial seed necessary to cause 50% mortality (LD50) of larvae was 3.6%, and that to reduce mass larvae by 50.0% (ED50) was 3.2%. Furthermore, the mass C. capitata larvae were affected at 53.2% and produced approximately 34% mortality at a level of 4.0% (w/w) of TTI incorporated in artificial diets.

Purification and characterization of a beta-glucuronidase present during embryogenesis of the mollusk Pomacea sp.

A beta-glucuronidase was purified from Pomacea sp. eggs by ammonium sulfate fractionation, DEAE-BioGel and Heparin-Sepharose chromatographies. This enzyme showed a Mr 180 kDa, with subunits of 90 kDa. The kinetic parameters were: pH 4.0, temperature 60 degrees C, Km 2.7 x 10(-6) and Vmax 15.3 microM/h, activator Mg+2, and inhibitor: lactone of D-saccharic acid. beta-glucuronidase is an exoglucuronidase involved in glycosaminoglycans metabolism with kinetics parameters similar to those found in mammals.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly).

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.

Alpha-amylase inhibitors from Ficus sp. seeds and their activities towards coleoptera insect pests.

Alpha-amylase Inhibitors were isolated from Ficus sp. (Gameleira) seeds by acetone fractionation and Sephadex G-50. Two inhibitors (alpha-PPAI and alpha-ZSAI) were tested against alpha-amylases from coleopteran larvae. alpha-PPAI was active to alpha-amylases of Callosobruchus maculatus (52%) and Zabrotes subfasciatus (53%). alpha-ZSAI was strongly active to Z. subfasciatus (100%) of and Mimosestes mimosae (98%). The alpha-ZSAI is a glycoprotein of approximately 50 kDa with an IC50 value of 0.074 microg microl(-1).

Cysteine proteinases and cystatins

This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction