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Characterization of a Soluble Phosphatidic Acid Phosphatase in Bitter Melon (Momordica charantia)

Author:
Cao, Heping, Sethumadhavan, Kandan, Grimm, Casey C., Ullah, Abul H.J.
Source:
PLosOne 2014 v.9 no.9 pp. 1-10
Subject:
Momordica charantia, acid phosphatase, assays, biosynthesis, cell membranes, centrifugation, cobalt, copper sulfate, cotyledons, cytoplasm, diacylglycerols, dry matter accumulation, enzymatic hydrolysis, fatty acids, ferrous sulfate, fruits, functional foods, gels, herbal medicines, magnesium, magnesium chloride, manganese sulfate, mass spectrometry, octoxynol, pH, phosphatidate phosphatase, phospholipids, proteins, seeds, sodium fluoride, taste, zinc sulfate
Abstract:
Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-snphosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg2+-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53–60uC and unaffected by up to 0.3 mM MgCl2. The Km and Vmax values for dioleoyl-phosphatidic acid were 595.4 mM and 104.9 gkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na3VO4, Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants.
Agid:
59645
Handle:
10113/59645