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Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of Momordica charantia

Journal: Advances in Biological Chemistry
ISSN 2162-2183

Volume: 03;
Issue: 01;
Start page: 11;
Date: 2013;
Original page

Keywords: PAP | EC | Phosphatidic Acid Phosphatase | Momordica Charantia

Phosphatidate phosphatase (EC, PAP, catalyzes the dephosphorylation of phosphatidate (PtdOH) to form diacylglycerol. In eukaryotes, PAP driven reaction is the committed step in the synthesis of triacylglycerol. A Mg2+ independent PAP activity was identified in the soluble extract of Momordica charantia cotyledons undergoing maturation. While the microsomal fraction of the extract gave only 10% of the PAP activity, the remaining 90% of the activity was associated with the soluble fraction. At pH 3.0, the soluble PAP was bound to S column and eluted with glycine-HCl buffer containing high salt. The pH and temperature optima of the PAP activity were 6.0 and 53℃, respectively. Under optimum assay condition, the Vmax and Km for dioleoyl phosphatidic acid were 1.89 ηkat/mg of protein and 142 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ- NPP, the Vmax and Km were 10.4 ηkat/mg of protein and 107 μM, respectively. The inclusion of Mg2+ and β-mercaptoethanol into the reaction mix did not change the enzyme activity nor did the addition of N-ethylmaleimide and phenylglyoxal, which indicates that cysteine and arginine are not involved in catalysis of PtdOH. The addition of Mg2+ up to 10 mM also did not change the level of PAP activity. Triton X-100, however, inhibited the activity. This is the first documented case of an in vitro PAP activity in the developing cotyledons of Momordica charantia. The PAP described here could serve as a model for lipin-1 or lipin-2 in humans. Mutations in these genes lead to acute myoglobinuria in human infants.
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