The effects of some compounds on exopolyphosphatase (A) and endopolyphosphatase (B, C) activities of purified PPN1 with PolyP₂₀₈ in the presence of 0.1 mM Co²⁺ or 0.25 mM Mg²⁺.

<p>The concentrations of effectors are (mM): P_i, PP_i, PolyP₃, ATP and ADP, 2.0; arginine, 100; spermidin and 1,3-diaminopropan, 50. The concentration of heparin was 0.01 mg/ml. Control, without effectors. A, white columns – exopolyphosphatase activity in the presence of Co²⁺, 100% corresponds to 290 U/ mg protein; black columns – exopolyphosphatase activity in the presence Mg²⁺, 100% corresponds to 22 U/mg protein. B, the endopolyphosphatase activity in the presence of Co²⁺; C, the endopolyphosphatase activity in the presence of Mg²⁺; - Me²⁺, without bivalent metal cation. The reaction time was 60 min. The PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. PolyP₂₀₈, the substrate was incubated without the enzyme for 60 min. The experiments were repeated in triple and the average values and typical photographs are shown.</p

The effect of ATP on the endopolyphosphatase reaction of PPN1 in the presence of 0.25 mM MgSO₄.

<p>PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. PolyP₂₀₈ - PolyP₂₀₈ was incubated without the enzyme at 30°C for 60 min. The numerals indicate ATP concentrations (mM). The PAGE experiment was repeated in triple and the photograph of typical experiment is shown.</p

Purification of recombinant PPN1 of <i>S. cerevisiae</i>.

<p>Purification of recombinant PPN1 of <i>S. cerevisiae</i>.</p

The SDS-PAGE of purified protein (A) and time dependence of PolyP₂₀₈ hydrolysis by the purified PPN1 (B-E).

<p>The protein markers (A) were phosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.1 kD), α-lactalbumin (14.4 kD). B, P_i release, % of P in PolyP₂₀₈ used as substrate; (○) in the presence of 0.1 mM CoSO₄, (●) in the presence of 0.25 mM of MgSO₄. C-E, PolyP PAGE was performed in 24% polyacrylamide gel with 7M urea; toluidine blue staining. C, PolyP markers, commercial PolyP with the average chain lengths of 15, 25, 45 and 65 phosphate residues from Sigma (USA) and PolyP with an average chain length of 208 phosphate residues from Monsanto (USA). D, hydrolysis products of PolyP₂₀₈ in the presence of 0.1 mM CoSO₄. E, hydrolysis products of PolyP₂₀₈ in the presence 0.25 mM of MgSO₄. Control, PolyP₂₀₈ was incubated without the enzyme at 30°C for 120 min. The experiments were repeated in triple and the average values and typical photograph are shown.</p