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Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions
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Pinilla, Yudi T.
Moreno-Pérez, Darwin A.
Patarroyo, Manuel A.
Bello, Felio J.
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2013
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Abstract
Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (N?-benzoyl- dl-Arg- p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ~69. kDa to ~23. kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ~25. kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. © 2013 Elsevier B.V.
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Keywords
Serine proteinase , Enzyme activity , Excretion , Fly , Larva , Secretion , Article , Controlled study , Diptera , Enzyme inhibition assay , Enzyme substrate , Hydrolysis , Larva , Nonhuman , Polyacrylamide gel electrophoresis , Protein degradation , Protein determination , Protein secretion , Sarconesiopsis magellanica , Species difference , Zymography , Calliphoridae , Diptera , Lucilia sericata , Larvae , Larval therapy , Necrotic wounds , Proteases , Proteolytic activity , S , Magellanica , Animals , Benzoylarginine nitroanilide , Bodily secretions , Chromogenic compounds , Diptera , Electrophoresis , Enzyme inhibitors , Humans , Hydrogen-ion concentration , Larva , Molecular weight , Peptide hydrolases , Proteolysis , Larvae , Larval therapy , Necrotic wounds , Proteases , Proteolytic activity , S , Magellanica
