Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom

Maruyama, Masugi; Sugiki, Masahiko; Yoshida, Etsuo; Mihara, Hisashi; Nakajima, Nobuyoshi

doi:10.1016/0041-0101(92)90383-g

Cited by 58 publications

(28 citation statements)

References 19 publications

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“…Finally, another source of trypsin-fold serine proteases is the Brazilian snake venom where the enzymes are involved in altering the victim's blood coagulation [17,33,34]. Therefore, to identify a thrombin-like enzyme on L. muta venom, a snake from the southeast of Brazil, a variant of ecotin was used, namely TSRR/R (V81T, T83S, M84R, M85R, and D70R) that is able to inhibit human α-thrombin.…”

Section: Resultsmentioning

confidence: 99%

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

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Section: Resultsmentioning

confidence: 99%

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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“…The venom also displayed moderate proteolytic activity. B. jararaca venom contains several well-described proteinases, such as jararagin (a 52 kDa hemorrhagic metalloproteinase), two fibrinolytic metalloproteinases (21 and 47 kDa, respectively), a 67-kDa trypsin-like serine proteinase, small hemorrhagins (w25 kDa), and others (Maruyama et al, 1992;Murayama et al, 2003;Paine et al, 1992). In our zymography analysis, we found that B. jararaca venom effected intense casein hydrolysis with bands ranging in size from 25 to 28 kDa.…”

Section: Discussionmentioning

confidence: 96%

In vitro comparison of enzymatic effects among Brazilian Bothrops spp. venoms

Campos

Pucca

Roncolato

et al. 2013

Toxicon

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In various types of snake venom, the major toxic components are proteinases and members of the phospholipase A2 family, although other enzymes also contribute to the toxicity. In this study, we evaluated the proteolytic, phospholipase, and L-Amino acid oxidase activities in the venom of five Bothrops species-Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, and Bothrops alternatus-all of which are used in the production of commercial antivenom, prepared in horses. The enzymatic activities of each species' venom were classified as high, moderate, or low. B. moojeni venom demonstrated the highest enzymatic activity profile, followed by the venom of B. neuwiedi, B. jararacussu, B. jararaca, and B. alternatus. To our knowledge, this is the first study to compare all of these enzymes from multiple species, which is significant in view of the activity of L-amino acid oxidase across Bothrops species.

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“…Several metalloproteinases have been isolated from Bothrops venoms [11,[19][20][21]. These zinc-dependent enzymes produce hemorrhage by hydrolyzing proteins in the basal lamina of capillary vessels [9,10,20,22] and their activity can be inhibited by metal chelators such as ethylene-diaminetetraacetic acid (EDTA) [23,24].…”

Section: Introductionmentioning

confidence: 99%