Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Abstract

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM-MgCl2, 0.5 mM-EDTA and 0.1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at -20 degrees C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3-208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, Km values were 1.7 x 10(-4), 1.5 x 10(-5) and 8.8 x 10(-7) M respectively. Polyphosphatase was most active and stable at pH 6.0-8.0. The enzyme showed maximal activity at 50 degrees C. The time of half inactivation of polyphosphatase at 40, 45 and 50 degrees C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca2+ or Cu2+, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co2+> Mg2+ > Mn2+ > Fe2+ > Zn2+. Polyphosphatase was completely inhibited by 1 mM-ammonium molybdate and 50 microM-Zn2+ or Cu2+ (in the presence of Mg2+).

Overexpression of a Zn2+-sensitive Soluble Exopolyphosphatase from Trypanosoma cruzi Depletes Polyphosphate and Affects Osmoregulation

“…5 (C and D). The rTcPPX activity, like in other exopolyphosphatases (17,19,(42)(43)(44)(45)(46)(47), was negligible in the presence of 5 mM EDTA and was stimulated in the presence of a divalent cation as metal cofactor. At low concentrations Mg 2ϩ , Mn 2ϩ , and Co 2ϩ (Ͻ1 mM) stimulated rTcPPX activity to different degrees, depending on the cation and substrate used.…”

Polyphosphatase PPX1 of <i>Saccharomyces cerevisiae</i> as a Tool for Polyphosphate Assay

“…The specific activities of the known preparations of purified PPX1 determined under the same conditions were 204 [24], 283 [31], and 202 U/mg protein [32]. Therefore, the preparation that we have obtained is one of the most active preparations described earlier.…”

“…The cases when other polyP and cations were used are indicated specially. PolyP (Monsanto, USA) was purified from pyrophosphate and orthophosphate as described previously [24]. The amount of the enzyme forming 1 μmol P i per minute was taken as a unit of enzyme activity (U).…”

“…Thus, the recombinant PPX1 preparation was similar in its physicochemical properties to the purified PPX1 preparations from the wild yeast strains [24] [31].…”

“…Specific enzymes hydrolyzing polyP are well known; among them, there is an exopolyphosphatase (polyphosphate phosphohydrolase EC 3.6.1.11) that splits P i from the end of the polyP chain. The yeast exopolyphosphatase PPX1 has a high specificity to polyP [24]- [26]. Recently, we have obtained a yeast strain with the overproduction of this enzyme [27].…”