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.
Highlights
Phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) hydrolyzes the phosphomonoester bond present in PtdOH yielding diacylglycerol (DAG) and Pi
A key step in the formation of TAG is the dephosphorylation of the sn-3 position of PtdOH, which is formed by the action of two specific acyltransferases, namely, glycerophosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT)
All three reagents had no effect on PAP activity (Table 1). This is the first reporting of any phosphatidic acid phosphohydrolase (EC 3.1.3.4) activity in Momordica charantia, which was chosen for study because the mature seeds of the plant make TAG that contains α-eleostearic acid as the acyl chains in all three positions [13]
Summary
Phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) hydrolyzes the phosphomonoester bond present in PtdOH yielding diacylglycerol (DAG) and Pi. In the cytoplasmic membranes of plant seed tissue that accumulate storage triglycerides (oil), fatty acyl groups are added sequentially by specific acyltransferase enzymes to the sn-1, sn-2, and sn-3 positions of glycerol-3-phosphate (G3P) to form triacylglycerol (TAG). This pathway was discovered in the 1950s and commonly referred to as the Kennedy or G3P pathway [1]. The discovery of lipin 1 being a PAP, the enzyme is linked to the regulation of body fat metabolism in mammalian cells It follows that the activity of PAP will be probed by pharmaceutical researchers for the control of body fat metabolism in humans
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