Identification of a soluble phosphatidate phosphatase (PAP) in the developing cotyledons of Gossypium hirsutum

Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate to form diacylglycerol.  In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG).   A Mg²⁺ independent PAP activity was identified in the soluble extract of Gossipium hirsutum maturing cotyledons.  While the microsomal fraction of the extract gave only 12% of the PAP activity, the remaining 88% of the activity was associated with the 105,000 x g supernate fraction.   The pH and temperature optima of the enzyme were 6.0 and 67 ºC, respectively. Under optimum assay condition, the V_max and K_m for dioleoyl phosphatidic acid (DPA) as susbtrate, were 2.8 ηkat/mg of protein and 539 µM, respectively.  The PAP activity correlated well with enzyme concentration and incubation time.  Inclusion of the detergent triton X-100 (0 - 0.5%) or Magnesium chloride (0 - 2 mM) in the reaction mix didn't alter activity to a significant degree.  This is the first report of an in vitro PAP activity in the developing cotyledons of Gossipium hirsutum