The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases.
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 the quantitative assay, 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 nmol h(-1)mg of protein(-1)) and L3 (7.7+/-0.1 nmol h(-1)mg of protein(-1)) and that both activities were partially inhibited by trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 15% and 3%, respectively. Also, we demonstrated that the Nalpha-p-Tosyl-l-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117+/-24 nmol h(-1)mg of protein(-1)) and L3 (111+/-10 nmol h(-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. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. These findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.
Pires
F A
FA
Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, CEP 21.045-900, Manguinhos, Rio de Janeiro, RJ, Brazil.
Moya-Borja
G E
GE
Barreira
J D
JD
Pinho
R T
RT
Alves
C R
CR
D000818:Animals; D004175:Diptera / Q000201:enzymology* / Q000254:growth & development; D019476:Insect Proteins / Q000378:metabolism*; D007814:Larva / Q000201:enzymology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism
D019476:Insect Proteins; D012697:Serine Endopeptidases