Title of article :
Elastase secretion in Acanthamoeba polyphaga
Author/Authors :
Ferreira، نويسنده , , Gabriela A. and Magliano، نويسنده , , Ana C.M. and Pral، نويسنده , , Elizabeth M.F. and Alfieri، نويسنده , , Silvia C.، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2009
Pages :
8
From page :
156
To page :
163
Abstract :
Acanthamoeba species are frequently isolated from soil and water collections. In the environment, the organisms multiply as phagotrophic trophozoites and encyst under adverse conditions. Several species are known to infect man, causing keratitis and opportunistic diseases. The mechanisms underlying tissue damage and invasion by the amoebae are being elucidated and the involvement of secreted peptidases, particularly serine peptidases, has been demonstrated. Here, elastase activity was examined in Acanthamoeba-conditioned medium (ACM), making use of elastin-Congo red (ECR) and synthetic peptide p-nitroanilide substrates. ACM hydrolysed ECR over a broad pH range and optimally at a pH of 7.5 and above. Indicating the activity of serine and metallopeptidases, Congo red release was potently inhibited by PMSF, antipain, chymostatin and 1,10-phenanthroline, partially reduced by elastatinal and EDTA, and unaffected by 1,7-phenanthroline and E-64. Screening with synthetic substrates mainly showed the activity of serine peptidases. ACM efficiently hydrolysed Suc-Ala2-Pro-Leu-pNA and Suc-Ala2-Pro-Phe-pNA over a broad pH range (7.0–9.5) and was weakly active against Suc-Ala3-pNA, a substrate found to be optimally hydrolysed at a pH around 7.0. Following ammonium sulfate precipitation of ACM proteins and FPLC analysis, the majority of the ECR-splitting activity, characterised as serine peptidases, bound to CM-sepharose and co-eluted with part of the Suc-Ala2-Pro-Phe-pNA-hydrolysing activity in a gradient of 0–0.6 M NaCl. In the corresponding FPLC fractions, serine peptidases resolving in the region of 70–130 kDa were detected in gelatin gels. Overall, the results demonstrate that trophozoites secrete elastases, and additionally suggest the high molecular weight serine peptidases as possible elastase candidates.
Keywords :
Acanthamoeba polyphaga , Elastases , Metallopeptidases , serine peptidases , Secretory activity , Acanthamoeba
Journal title :
Acta Tropica
Serial Year :
2009
Journal title :
Acta Tropica
Record number :
1740666
Link To Document :
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