Purification, Characterization, and Bioinformatics Studies of Phosphatidic Acid Phosphohydrolase from Lagenaria siceraria
2012
Ullah, Abul H.J. | Sethumadhavan, Kandan | Grimm, Casey | Shockey, Jay
Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacylglycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5C and 45C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 kat/mg of protein and 200 μM, respectively. For the synthetic sub-Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of trypsinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.
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