Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform F. A. REZENDE-SOARES 1 , C. CARVALHO-CAMPOS 1 , M. J. MARQUES 1,2 , G. N. PORCINO 1 , N. L. L. GIAROLA 1 , B. L. S. COSTA 1 , A. TAUNAY-RODRIGUES 1 , P. FARIA-PINTO 1 , M. A. SOUZA 1,3 , V. A. DINIZ 4 , S. CORTE-REAL 4 , M. A. JULIANO 5 , L. JULIANO 5 and E. G. VASCONCELOS 1 * 1 Departamento de Bioquı ´mica/Po´s-Graduac¸a˜ o em Imunologia, Gene´tica e Biotecnologia/ICB, Universidade Federal de Juiz de Fora, Campus Universita´rio, Bairro Cidade Universita´ria, Juiz de Fora, MG, Brazil 2 Departamento de Cieˆncias Biolo´gicas, Universidade Federal de Alfenas, Alfenas, MG, Brazil 3 Laborato´rio de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberlaˆndia, Uberlaˆndia, MG, Brazil 4 Laborato´rio de Biologia Estrutural, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil 5 Departamento de Biofı ´sica, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil (Received 8 April 2009; revised 15 September and 16 October 2009; accepted 20 October 2009; first published online 7 December 2009) SUMMARY An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83 % ATPase and 87 % of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1 : 50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80 % seropositivity) and its potato apyrase counterpart (50 % seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response. Key words : ATP diphosphohydrolase, potato apyrase, NDPase, GDPase, ecto-enzyme, Leishmania (Viannia) braziliensis, promastigote, American cutaneous leishmaniasis. INTRODUCTION Leishmanias are digenetic protozoan parasites that live as promastigotes in the digestive tract of sandflies and as amastigotes in the phagolysosomes of mam- malian macrophages. At least 20 species of Leish- mania are known to infect mammals, causing a wide range of clinical manifestations determined by the parasite species, host genetic and immune factors (Requena et al. 2000; Gonc¸alves-Da-Costa, 2005 ; Kedzierski et al. 2006). The genes in L. braziliensis, L. infantum and L. major genomes are being in- vestigated to determine their roles in establishment of infection or involvement as virulent factors, and in parasite survival (Peacock et al. 2007; Smith et al. 2007). Putative proteins identified as nucleoside diphos- phatases (NDPases) and guanosine diphosphatases (GDPases), homologous to the members of the ATP diphosphohydrolase family, were found in the genomes of L. major, L. infantum and L. braziliensis parasites (Peacock et al. 2007). ATP diphospho- hydrolases (EC 3.6.1.5), also known as apyrases, were found in distinct organisms, and share several common features, such as ability to hydrolyse nucleosides di- and triphosphates to the corres- ponding nucleoside monophosphates upon bivalent metal ion activation. In pathogenic agents such as Leishmania amazonensis, Schistosoma mansoni, Trichomonas vaginalis, Taenia crassiceps cysticerci and Legionella pneumophila, this protein has been described as an ecto-enzyme (Coimbra et al. 2002; Faria-Pinto et al. 2004, 2006 ; Pinheiro et al. 2006; * Corresponding author : Departamento de Bioquı ´mica, ICB, Universidade Federal de Juiz de Fora, Campus Universita´rio, Bairro Cidade Universita´ria, 36036-330, Juiz de Fora, MG, Brazil. Tel: +55 32 2102 3217. Fax: +55 32 2102 3216. E-mail : eveline.vasconcelos@ ufjf.edu.br 773 Parasitology (2010), 137, 773–783. f Cambridge University Press 2009 doi:10.1017/S0031182009991661