Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BA rho NA, Michaelis-Menten behavior with K(m) 2.3 x 10(-1) M and the V(max) 0.52 x 10(-1) nmoles rho-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Set, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause alpha- and beta-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity. (C) 2009 Elsevier Ltd. All rights reserved.554745753Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ms Sc thesis of Augusto Vilca Quispe [06/54275-7]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ms Sc thesis of Augusto Vilca Quispe [06/54275-7

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function. (C) 2007 Elsevier Inc. All rights reserved.358385486

Biochemical, pharmacological and structural characterization of two PLA(2) isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom

Cdr-12 and Cdr-13 isoforms of PLA(2), a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on mu-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA(2) was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA(2). The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13. This sequence shows high identity values when compared to other D49 PLA(2)s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA(2) isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA(2), caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA(2) family, which presents the typical bioactivities described for such proteins.261394

Xylella fastidiosa disturbs nitrogen metabolism and causes a stress response in sweet orange Citrus sinensis cv. Pera

Xylella fastidiosa (Xf) is a fastidious bacterium that grows exclusively in the xylem of several important crop species, including grape and sweet orange (Citrus sinensis L. Osb.) causing Pierce disease and citrus variegated chlorosis (CVC), respectively. The aim of this work was to study the nitrogen metabolism of a highly susceptible variety of sweet orange cv. 'Pera' (C. sinensis L. Osbeck) infected with Xf. Plants were artificially infected and maintained in the greenhouse until they have developed clear disease symptoms. The content of nitrogen compounds and enzymes of the nitrogen metabolism and proteases in the xylem sap and leaves of diseased (DP) and uninfected healthy (HP) plants was studied. The activity of nitrate reductase in leaves did not change in DP, however, the activity of glutamine synthetase was significantly higher in these leaves. Although amino acid concentration was slightly higher in the xylem sap of DP, the level dropped drastically in the leaves. The protein contents were lower in the sap and in leaves of DP. DP and HP showed the same amino acid pro.les, but different proportions were observed among them, mainly for asparagine, glutamine, and arginine. The polyamine putrescine was found in high concentrations only in DP. Protease activity was higher in leaves of DP while, in the xylem sap, activity was detected only in DP. Bidimensional electrophoresis showed a marked change in the protein pattern in DP. Five differentially expressed proteins were identified (2 from HP and 3 from DP), but none showed similarity with the genomic (translated) and proteomic database of Xf, but do show similarity with the proteins thaumatin, mucin, peroxidase, ABC-transporter, and strictosidine synthase. These results showed that significant changes take place in the nitrogen metabolism of DP, probably as a response to the alterations in the absorption, assimilation and distribution of N in the plant.58112733274

Biochemical, Pharmacological, and Structural Characterization of New Basic PLA(2) Bbil-TX from Bothriopsis bilineata Snake Venom

Bbil-TX, a PLA(2), was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on mu-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by -Tof ltima API ESI/ MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA(2) from snakes and shows high identity values when compared to other PLA(2)s. PLA(2) activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37 degrees C. Maximum PLA(2) activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The infiammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinfiammatory effect, the phospholipid hydrolysis may be relevant for these phenomena

Isolation and biochemical characterization of a galactoside binding lectin from Bauhinia variegata candida (BvcL) seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized D-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.26319320