Partial Purification and Immunohistochemical Localization of ATP Diphosphohydrolase from Schistosoma mansoni

Abstract

ATP diphosphohydrolase from tegumental mem- branes of Schistosoma mansoni was solubilized with Tri- ton X-100 plus deoxycholate and separated by prepara- tive nondenaturing polyacrylamide gel electrophoresis. Two isoforms with ATP-hydrolytic activity were identi- fied and excised from nondenaturing gels. For each of the active bands, two protein bands (63 and 55 kDa) were detected with Coomassie Blue staining, following sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. Western blots developed with polyclonal anti- potato apyrase antibody revealed a single protein of 63 kDa, either with samples excised from active bands or with total S. mansoni tegument. Anti-potato apyrase an- tibody immobilized on Sepharose-Protein A depleted over 95% of ATPase and ADPase activities from deter- gent-solubilized tegument. Confocal laser scanning mi- croscopy showed anti-potato apyrase antibody on the outer surface of S. mansoni tegument. A different anti- body against a fusion protein derived from recently cloned Toxoplasma gondii nucleoside triphosphate hy- drolase (Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252–29260) revealed the same 63-kDa band in Western blots of S. mansoni tegument. Since anti-potato apyrase antibodies exhibited cross-reactivity with S. mansoni ATP diphosphohydrolase, we decided to gain further information on the primary structure of potato apyrase by sequencing the protein. Three novel peptides were obtained: amino-terminal sequence and two internal se- quences from tryptic fragments. Eight sequences re- cently deposited in the data bank, including that of T. gondii nucleoside triphosphate hydrolase, have consid- erable homologies to potato apyrase suggesting a new family of nucleoside triphosphatases which contains a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino-terminal. Antibody cross-reactivities in the pres- ent work suggest that conserved epitopes from S. man- soni ATP diphosphohydrolase are present in this family of nucleotide-splitting enzymes.