Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope.
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+).
Andreeva
N A
NA
Laboratory of Biochemistry of Membrane Processes, Russian Academy of Sciences, Pushchino, Moscow Region.
Okorokov
L A
LA
D017766:Acid Anhydride Hydrolases; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010743:Phospholipids / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D002413:Cations, Divalent; D010743:Phospholipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase