Boophilus microplus tick larvae‚ a rich source of Kunitz type serine proteinase inhibitors

Autor(es)

Sasaki‚ S. D. ; Azzolini‚ S. S. A. ; Hirata‚ I. Y. ; Andreotti‚ R. ; Tanaka‚ A. S.

• Tipo de documento: Artigo Científico
• Fonte: Biochimie; v. 86, n. 9/10, p. 643-649, Sept.-Oct. 2004.
• Área do conhecimento: Ciências Biológicas - Bioquímica
• Assunto(s): Enzimas Peptídeo hidrolases Carrapatos Boophilus microplus

Resumo

Serine proteinase inhibitors from Boophilus microplus tick larvae (BmTIs) were purified by affinity chromatography on a trypsin-Sepharose column. BmTIs presented molecular weight between Mr 6200 and 18,400 and inhibitory activity for trypsin, HuPK (human plasma kallikrein) and neutrophil elastase. Using ion exchange chromatography, BmTIs were separated in several protein pools named BmTI-A to BmTI-F and BmTI-1 to BmTI-7. All BmTI forms presented inhibitory activity for trypsin with apparent dissociation constants (Ki) in the nM range. In this work, we describe the purification of BmTI-D, BmTI-2, and BmTI-3. These three inhibitors affected neutrophil elastase and HuPK with Ki also in nM range. BmTI-D proved to be the best HuPK inhibitor, while BmTI-3 was more efficient for neutrophil elastase with dissociation constants (Ki) of 12 and 0.5 nM, respectively. BmTI-D, BmTI-2, and BmTI-3 N-terminal amino acid sequences allowed us to include them into the BPTI-Kunitz type serine proteinase inhibitor family. BmTIs purified on trypsin-Sepharose were also used in a bovine immunization assay, resulting in antibody (anti-BmTIs) production. (AU)

• Processo FAPESP: 99/09097-8 - Estudos moleculares de substancias anti-hemostaticas presentes em artropodes hematofagos.
• Beneficiário: Aparecida Sadae Tanaka
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 99/12192-2 - Isolamento e caracterização de inibidores de serinoproteases (BmTIs) presentes em larvas de carrapatos Boophilus microplus: clonagem e sequenciamento do gene de um inibidor (BmTI)
• Beneficiário: Sergio Daishi Sasaki
• Modalidade de apoio: Bolsas no Brasil - Mestrado