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

Abstract

ABSTRACT. Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55°C. The rAsPPase was heat stable at 40°C in the absence of added Mg 2+ and at 50°C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg 2+ > Co 2+ > Cu 2+ > Fe 2+ > Zn 2+ >Mn 2+ . On the contrary, enzyme activity was inhibited by Ca 2+ . These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms. KEY WORDS: Ascaris suum, enzyme activity, Family I inorganic pyrophosphatase.J. Vet. Med. Sci. 66(2): 221-223, 2004 Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an important enzyme that hydrolyzes inorganic pyrophosphate (PP i ) to produce 2 inorganic ortho-phosphates (P i ). The major role of PPase is to control the level of PP i which have been continuously produced as a result of biosynthesis of macromolecular compounds in the living cell. The enzyme is shown to be essential for life [3,9]. There are several categories of PPases, of which soluble PPases identified from prokaryote, yeast, plant and mammalian tissues, have been classified into Family I and Family II PPases to date. The two families of PPases, however, have no sequence similarity at amino acid level and have distinct catalytic features [10,13,14,19]. 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. The enzymes were intensely localized under the hypodermis of adult worm. Interestingly, we demonstrated a novel role of PPase enzyme in the development and molting process of A. suum third-stage larvae to fourth-stage in vitro. It is therefore of interest to investigate into the enzymatic properties of molting-associated AsPPase in some details. In this study, we determined temperature dependence, heat stability and divalent metal ions-dependent activity of A. suum PPase enzyme to provide insight into the PPase-regulated roundworm metabolism and/or optimum growth and survival.Recombinant AsPPase with a specific activity of 937 µmol P i /min/mg of protein was used in the present study. 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 m...

Functional Characteristics of Inorganic Pyrophosphatase from Psychrotroph Shewanella sp. AS-11 upon Activation by Various Divalent Cations

“…Both families of inorganic pyrophosphatases are only active in the presence of metal ion cofactors, which perform numerous functions in catalysis, but differ in their catalytic properties and structure. Family I inorganic pyrophosphatases show strong metal ion dependency, with Mg 2+ ions providing the highest inorganic pyrophosphate hydrolysis activity 13 . Family II inorganic pyrophosphatases are more active with Mn 2+ or Co 2+ ions as cofactors instead of Mg 2+ ions 11,14 ions confer a 20-fold higher activity to family II inorganic pyrophosphatases than do Mg 2+ ions 15 .…”