Purification and Partial Characterization of an Exopolyphosphatase in Boophilus microplus Eggs
Evenilton P. Costa1; Eldo Campos1; Arnoldo R. Façanha1; Aoi Masuda2; Itabajara Vaz Jr.2 and Carlos Logullo1
1) Laboratório de Química e Função de Proteínas e Peptídeos and Laboratório de Biologia Celular e Tecidual – CBB – UENF, Campos dos Goytacazes, RJ, Brazil. 2) Centro de Biotecnologia / UFRGS, Porto Alegre, RS, Brazil.
The cattle tick Boophilus microplus is one of the most important ectoparasites of livestock, both in Brazil and in the tropics and subtropics in general. According to the Brazilian Agriculture Ministry B. microplus is responsible for losses in the order of one billion dollars a year. In Brazil, this parasite causes 65% of direct damage and 35% of indirect damage to cattle. Metabolic studies of tick metabolism can be an important tool for the understanding and/or control of its biological dynamics.
Polyphosphates (PolyP) are linear polymers of orthophosphate linked by an energy-rich phosphoanhydride bond, serving simultaneously as phosphate and energy reserves of the cell. They have been found in almost all living beings. These compounds are highly mobile cell components and in certain conditions, polyPs are able to be involved in metabolic processes. The exopolyphosphatase is an important enzyme to development of tick and play important role of hydrolyze chains of polyphosphate to Pi.
1g of eggs was homogenized in 10mL of 10mM Tris–HCl pH 7.4, containing 100µM leupeptin and 100 nM pepstatin. The fraction 1 was supplemented with 10% saturated of (NH4)2SO4 and the pellet was removed by centrifugation and dissolved in 10mL of 10mM Tris-HCl (pH 7.4). The fraction 2 was adjusted to 15% saturated of (NH4)2SO4 and applied to a phenyl-Sepharose column, equilibrated with 10mM Tris-HCl (pH 7.4), containing 15% of (NH4)2SO4. After equilibrated with the same buffer, the column was eluted with a linear gradient from 15% to 0% of (NH4)2SO4. Fraction containing the major exopolyphosphatase activity was colleted and applied on the Superose 6 column equilibrated with 10mM Tris-HCl pH 7.4, contained 150mM NaCl. Partial exopolyphosphatase purified was monitored by electrophorese 10% of polyacrilamide. We have been analyzed the Km, Ki, and the optimum pH of this enzyme.
These results will be contributing to understand the general metabolism during the B. microplus embryogenesis.
Supported by PRONEX, FAPERJ, CNPq and CAPES.
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