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Previous issue date: 2007-04-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / We performed a combination of proteinase assay, either in solution or immobilized in sodium
dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify
proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In quantitative
assays, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for
the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was
hydrolyzed by crude extracts of L2 (3.0 ? 0.2 nmoles hour-1 mg of protein-1) and L3 (7.7 ? 0.1
nmoles hour-1 mg of protein-1) and that both activities were partially inhibited by transepoxysuccinyl-
L-leucylamido-(4-guanidino)butane, 15 % and 3 % respectively. Also, we
demonstrated that the Na-p-Tosyl-L-Arg methyl ester substrate was hydrolyzed by crude
extracts of L2 (117 ? 24 nmoles hour-1 mg of protein-1) and L3 (111 ? 10 nmoles hour-1 mg of
protein-1), suggesting a predominance of esterase activity in the crude larval preparation.
Interestingly, the specific activity of serine-proteinases was totally inhibited by
Phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10 % of this enzyme class
activity was inhibited in the L2 crude extract. Also, we have detected crude extract L2 (Km =
7,59) larvae have more affinity than L3 larvae (Km = 35,75) to Na-p-Tosyl-L-Arg methyl
ester. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae
express serine-proteinases with similar (13 kDa and 22 kDa) and distinct (50 kDa in L2 and
30 kDa in L3) relative molecular masses. Additionally, we have isolated an enriched esterase
activity from L3 crude extract using successive chromatographies in Aprotinine-Agarose and
DEAE-Sephacell columns. By this strategy we detected only one 50 kDa proteinase in this
larvae crude extract. Finally, these findings contribute to the biochemical characterization of
D. hominis L2 and L3 larvae. / Neste trabalho foram realizados ensaios de atividade de proteinase em solu??o e com
prote?nas imobilizadas em gel de poliacrilamida contendo dodecil sulfato de s?dio
copolimerizado com gelatina, para detec??o e quantifica??o das proteinases presentes nos
extratos larvares de segundo (L2) e terceiro (L3) est?gios de Dermatobia hominis. Nos
ensaios quantitativos, utilizou-se um painel de pept?deos sint?ticos espec?ficos para as
principais classes de proteinases. Verificamos que o substrato pGlu-Phe-Leu p-nitroanilide foi
hidrolisado pelo extrato total de L2 (3,0 ? 0,2 nmoles hora-1 mg de prote?na-1) e L3 (7,7 ? 0,1
nmoles hora-1 mg de prote?na-1) e que ambas atividades foram parcialmente inibidas pelo
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, 15 % e 3 % respectivamente.
Tamb?m, demonstramos que o substrato Na-p-Tosyl-L-Arg methyl ester foi hidrolisado pelos
extratos totais de L2 (117 ? 24 nmoles hora-1 mg de prote?na-1) e L3 (111 ? 10 nmoles hora-1
mg de proteina-1), sugerindo uma predomin?ncia da atividade ester?sica nestes extratos. A
atividade espec?fica de serino-proteinases foi totalmente inibida pelo phenylmethylsulphonyl
fluoride nos extratos de L3, enquanto que somente 10 % desta atividade foi inibida nos
extrados de L2. Al?m disso, n?s detectamos que o extrato total das larvas L2 (Km = 7,59) tem
maior afinidade ao Na-p-Tosyl-L-Arg methyl ester do que o extrato total das larva de L3 (Km
= 35,75). Os resultados do ensaio qualitativo com g?is de substrato sugerem que os extratos
larvares L2 e L3 expressam serino-proteinases com similares (13 kDa e 22 kDa) e distintas
(50 kDa em L2 e 30 kDa em L3) massas moleculares relativas. Adicionalmente, isolamos
uma atividade ester?sica enriquecida do extrato total de L3 utilizando sucessivas
cromatografias em colunas de Aprotinina-Agarose e DEAE-Sephacell. Com esta estrat?gia,
detectamos somente uma banda de proteinase de 50 kDa neste extrato total. Estes resultados
contribuem para a caracteriza??o das proteinases larvares de D. hominis.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:tede/815 |
| Date | 26 April 2007 |
| Creators | Pires, Fabiano Araujo |
| Contributors | Borja, Gonzalo Efrain Moya, Alves, Carlos Roberto, Barreira, Jairo Dias |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Curso de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Parasitologia Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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