Partial purification and properties of phosphatidate phosphatase in Saccharomyces cerevisiae.
Using an aqueous dispersion of [32P]phosphatidate as substrate we detected phosphatidate phosphatase (EC 3.1.3.4) activity in a cell-free extract of the yeast, Saccharomyces cerevisiae. The activity was found in both the membrane and the soluble fractions. The enzyme was purified from the soluble fraction about 600-fold. The purification procedure involved (NH4)2SO4 fractionation, poly(ethylene glycol) 6000 fractionation and column chromatography on DEAE-Sepharose, Sephadex G-100 and Blue-Sepharose. The purified enzyme almost absolutely required Mg2+ for activity. The molecular weight of the enzyme was estimated by analytical gel filtration on Sephadex G-100 to be approx. 75000. The enzyme was highly specific for phosphatidate. The apparent Km for phosphatidate was approx. 0.05 mM. The optimum pH was between 7.0 and 8.0.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D010761:Phosphorus Radioisotopes; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase