Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of Momordica charantia ()
ABSTRACT
Phosphatidate phosphatase (EC 3.1.3.4), 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.
Share and Cite:
Ullah, A. and Sethumadhavan, K. (2013) Identification of a soluble phosphatidate phosphohydrolase in the developing cotyledons of
Momordica charantia.
Advances in Biological Chemistry,
3, 11-17. doi:
10.4236/abc.2013.31002.