Purification and Characterization of a Soluble Polyphosphatase from Mitochondria of Saccharomyces cerevisiae

L. P. Lichko^*, T. V. Kulakovskaya, and I. S. Kulaev

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-095) 923-3602; E-mail: alla@ibpm.serpukhov.su

^* To whom correspondence should be addressed.

Received July 26, 1999

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A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of ~41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is ~2-fold higher with tripolyphosphate. ATP, PP_i, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent K_m values are 300, 18, and 0.25 µM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg²⁺ = Co²⁺ = Mn²⁺ > Zn²⁺). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.

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KEY WORDS: polyphosphatase, mitochondria, purification, molecular and kinetic properties, Saccharomyces cerevisiae

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