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 approximately 125 kD. A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, 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 polyP3 is 13% of that with polyP208. The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively. The enzyme is most active at pH approximately 7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold. The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.
Andreeva
N A
NA
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia. alla@ibpm.pushchino.ru
Kulakovskaya
T V
TV
Kulaev
I S
IS
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004791:Enzyme Inhibitors; D017766:Acid Anhydride Hydrolases; C020137:exopolyphosphatase