Inorganic pyrophosphatase in the roundworm Ascaris and its role in the development and molting process of the larval stage parasites

Abstract

Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho‐phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli‐expressed recombinant enzyme has a specific activity of 937 µmol Pi·min−1·mg−1 protein corresponding to a kcat value of 638 s−1 at 55 °C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose‐dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble‐ and membrane‐bound H+‐PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.

Temperature and Metal Ions-Dependent Activity of the Family I Inorganic Pyrophosphatase from the Swine Roundworm Ascaris suum

“…However, the reason for increasing heat stability in the presence of Mg 2+ is still unclear. In the previous study, we showed that the rAsPPase absolutely required Mg 2+ for PP i hydrolysis [7]. The maximum enzyme activity was observed with 5 mM Mg 2+ , which then gradually declined with the increasing concentrations of Mg 2+ .…”

“…Among the Family I PPases, the most well characterized PPases are those from E. coli [8,12] and yeast [4,11,15]. The enzymes have shown strong metal iondependency, with Mg 2+ conferring the highest PP i -hydrolysis activity [4], and sensitive to inhibition by Ca 2+ [17].Recently, we cloned and characterized a gene that encodes a functional Family I PPase of the swine roundworm A. suum [7]. The A. suum PPase (AsPPase) has a molecular mass of 40 kDa and a pI of 7.1, and its homologs were expressed in human and dog roundworms, A. lumbricoides and Toxocara canis respectively.…”

“…Recently, we cloned and characterized a gene that encodes a functional Family I PPase of the swine roundworm A. suum [7]. The A. suum PPase (AsPPase) has a molecular mass of 40 kDa and a pI of 7.1, and its homologs were expressed in human and dog roundworms, A. lumbricoides and Toxocara canis respectively.…”

“…Enzyme activity was determined spectrophotometrically using a molybdate-blue based colorimetric assay as previously described [7]. Enzyme (specific) activity was defined as µmol of P i liberated by the hydrolysis of PP i per min per mg of protein.…”

“…The residual activity of the enzyme was then assessed. In the previous study we determined the optimum concentrations of Mg 2+ for enzyme activity [7]. In this study we examined whether other divalent metal ions (Co 2+ , Cu 2+ , Fe 2+ , Zn 2+ , Mn 2+ , Ca 2+ ) in order to determine the preferences for metal ions.…”