ATPases - Structure and Function

• Grant number: 95/09467-9
• Support Opportunities: Research Projects - Thematic Grants
• Start date: July 01, 1996
• End date: February 28, 2002
• Field of knowledge: Biological Sciences - Morphology
• Principal Investigator: Sergio Verjovski Almeida
• Grantee: Sergio Verjovski Almeida
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Associated research grant(s): 97/05780-0 - Destabilization of mrna coding for phosphorylation-site mutants of a rabbit ca2+atpase expressed in yeast., AR.EXT; 96/06571-2 - Partial purification and immunohistochemical localization of ATP diphosphohydrolase from S. mansoni, PUB.ART; 96/06812-0 - Heterologous expression of sarcoplasmic reticulum Ca2+-ATPase, PUB.ART; 96/04906-7 - Partial purification of atp-diphosphohydrolase from schistosoma mansoni - immunological cross reactivities with potato apyrase and t.gondii nucleoside triphosphate hydrolase., AR.EXT

Abstract

Genetic engineering is used as a tool for the study of rabbit muscle Calcium-ATPase structure and its relation to function. We have obtained in yeast the expression of wild-type rabbit sarcoplasmic reticulum Calcium-ATPase and of point mutants, in addition to a tagged Calcium-ATPase containing an amino-terminal hexa-histidine sequence which was introduced to facilitate purification. The hydrophilic domains of Calcium-ATPase have been expressed in bacteria and successfully purified. Spectroscopic measurements by CD and fluorescence are used to characterize the folding and ligand stabilization of these hydrophilic domains. The final goal of the project is to understand at the molecular level the mechanism of energy conversion involved in ion transport catalyzed by this enzyme.Another membrane protein that we are working with is the ATP-diphosphohydrolase (or apyrase) from the external surface of Schistosoma mansoni, a novel enzyme that we have characterized in 1993 andwhich belongs to a new family of ecto-ATPases that we have identified in 1996. Presently we are on the way of obtaining the clone for S. mansoni ecto-ATPase. The objective is to obtain its primary sequence and toidentify functional domains. At present we postulate that the enzyme may participate in the escape mechanisms of the parasite by degrading ADP on its external surface. This enzyme might be a target for drugs or a vaccine; in the long run the objective of our work is to establish the role of theenzyme in the interaction human host-parasite and to possibly explore its suitability as a target for treatment of infected humans. (AU)