Phosphatidate phosphatase (EC 126.96.36.199), 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.
Cite this paper
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