Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid(substrate)and diacylglycerol(product),which are lipid precursors used for the synthesis ...Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid(substrate)and diacylglycerol(product),which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol.Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression,membrane expansion,secretion,and endocytosis.While this important enzyme has been known for several decades,its gene was only identified recently from yeast.This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.展开更多
Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understan...Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.展开更多
基金supported in part by National Institutes of Health Grant GM-28140.
文摘Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid(substrate)and diacylglycerol(product),which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol.Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression,membrane expansion,secretion,and endocytosis.While this important enzyme has been known for several decades,its gene was only identified recently from yeast.This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.
文摘Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.
