Abstract
Phosphatidate phosphatase [EC 3. 1. 3. 4] was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfate precipitation, and molecular-sieve chroma-tography. For the enzyme assay, an aqueous dispersion of phosphatidate, rather than “membrane-bound” phosphatidate, was used as substrate.
The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Moreover, the activity of the enzyme is stimulated by phosphatidyl-choline. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuri-benzoate.
The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidyl-choline. In contrast to the results of previous studies, most of the enzyme activity was found in the soluble fraction.
