A Matin1, N A Khan. 1. School of Biological and Chemical Sciences, Birkbeck, University of London, London, England, UK.
Abstract
AIMS: To investigate the presence and partial characterization of ecto-ATPase in Balamuthia mandrillaris. METHODS AND RESULTS: In vitro assays were used to demonstrate that live B. mandrillaris hydrolyses extracellular AtP. Using nondenaturing polyacrylamide gel electrophoresis, B. mandrillaris exhibited a single ecto-ATPase band of molecular mass of more than 545 kDa. This ecto-ATPase was insensitive to ouabain, levamisole, sodium azide and sodium orthovanadate but stimulated by MgCl2. The ecto-ATPase was heat stable, but labile to detergent, sodium dodecyl sulphate. Suramin, an antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, inhibited B. mandrillaris binding to and cytotoxicity of HBMEC (human brain microvascular endothelial cello), in vitro. CONCLUSIONS: For the first time, we describe that live B. mandrillaris hydrolyses extracellular ATP and exhibits a > 545kDa ecto-ATPase. SIGNIFICANCE AND IMPACT OF THE STUDY: This surface enzyme may play a role in the salvage of purines from the extracellular medium and may be important for the pathogenesis of B. mandrillaris.
AIMS: To investigate the presence and partial characterization of ecto-ATPase in Balamuthia mandrillaris. METHODS AND RESULTS: In vitro assays were used to demonstrate that live B. mandrillaris hydrolyses extracellular AtP. Using nondenaturing polyacrylamide gel electrophoresis, B. mandrillaris exhibited a single ecto-ATPase band of molecular mass of more than 545 kDa. This ecto-ATPase was insensitive to ouabain, levamisole, sodium azide and sodium orthovanadate but stimulated by MgCl2. The ecto-ATPase was heat stable, but labile to detergent, sodium dodecyl sulphate. Suramin, an antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, inhibited B. mandrillaris binding to and cytotoxicity of HBMEC (human brain microvascular endothelial cello), in vitro. CONCLUSIONS: For the first time, we describe that live B. mandrillaris hydrolyses extracellular ATP and exhibits a > 545kDa ecto-ATPase. SIGNIFICANCE AND IMPACT OF THE STUDY: This surface enzyme may play a role in the salvage of purines from the extracellular medium and may be important for the pathogenesis of B. mandrillaris.
Authors: Pauline M Leite; Rodrigo S Gomes; Amanda B Figueiredo; Tiago D Serafim; Wagner L Tafuri; Carolina C de Souza; Sandra A L Moura; Juliana L R Fietto; Maria N Melo; Fátima Ribeiro-Dias; Milton A P Oliveira; Ana Rabello; Luís C C Afonso Journal: PLoS Negl Trop Dis Date: 2012-10-11