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-sn-phosphatidate 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 53–60°C and unaffected by up to 0.3 mM MgCl2. The Km and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/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.
Highlights
Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste
As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, we focused our studies on Phosphatidic acid phosphatases (PAPs) (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that dephosphorylates phosphatidic acid (PA, called PtdOH) to generate DAG and Pi
Following dialysis and centrifugation of the supernatant, the S3-pellet contained only 1.8% whereas the S3-supernatant contained 87.2% of the total activity (Figure 2). These subcellular distributions of PAP activity clearly demonstrated that the great majority of PAP activity in bitter melon seeds was soluble and localized in the cytosol
Summary
Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. It is a tropical and subtropical vine of the Cucurbitaceae family and widely grown in Asia, Africa and the Caribbean. The fruit has a distinct warty exterior and an oblong shape It is hollow in cross-section with a relatively thin layer of flesh surrounding a central seed cavity filled with large, flat seeds and pith. Bitter melon is often used in Chinese cooking for its bitter flavor, typically in stir-fries, soups and herbal teas It has been used as the bitter ingredient in some Chinese and Okinawan beers. The fatty acid compositions of bitter melon oil include 37% of saturated fatty acids mainly stearic acid; 3% of monounsaturated fatty acid dominantly linoleic acid, and 60% of polyunsaturated fatty acid predominately alpha-eleostearic acid (a-ESA, 9cis, 11trans, 13trans octadecatrienoic acid) which counts for 54% of the total fatty acids [1]
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