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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Proteinases Larvares de Dermatobia hominis (Linnaeus Jr., 1781) (DIPTERA: CUTEREBRIDAE). / Dermatobia hominis (Linnaeus Jr., 1781) (DIPTERA: CUTEREBRIDAE) Larvae Proteinases.

Pires, Fabiano Araujo 26 April 2007 (has links)
Made available in DSpace on 2016-04-28T20:16:24Z (GMT). No. of bitstreams: 1 2007- Fabiano Araujo Pires-01.pdf: 878766 bytes, checksum: 9179bfb28ddd6cee14d8d4414534b8f8 (MD5) 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.

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