Purification and properties of exopolyphosphatase from the cytosol of Saccharomyces cerevisiae not encoded by the PPX1 gene

A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium. The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it. Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9%. The minimal molecular mass of the active but unstable enzyme complex is similar to125 kD. A stable enzyme complex with a molecular mass of similar to500 kD is composed of two polypeptides of similar to32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with PolyP(3), not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent. The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate. The activity with polylP(3) is 13% of that with polyP(208). The K-m values for polyP(208), polyP(15), and polyP(3) hydrolysis are 3.5, 75, and 1100 muM, respectively. The enzyme is most active at pH similar to7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of I in M enhances it 2-fold. The enzyme understudy is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.