| Summary: | 碩士 === 國立臺灣大學 === 植物科學研究所 === 95 === Degradation and mobilization of reserve food in germinating seeds are essential processes for establishing a healthy seedling. Lipase is the crucial enzyme responsible for hydrolysis of triacyglycerol into fatty acids leading the conversion of lipid to carbohydrate. Proteinaceous inhibitors of lipase have been demonstrated in many plant species. Lipoxygenase isolated from germinating soybean cotyledon can compete for lipid substrate and show the inhibitory effect on pancreatic lipase in vitro. Satouchi et al. (2002) reported that a protein existing in lipoxygenase-deficient soybean seeds also acted as lipase inhibitor. The inhibitor was further identified as a beta-amylase by MALDI-MS analysis. In a germinating maize kernel, beta-amylase activity is synthesized in aleurone cells where no starch exists, and unlike beta–amylase, beta–amylase is not secreted to starchy endosperm. Owing to the inaccessibility of substrate, the physiological function of aleurone beta-amylase is unknown. Some lipases reported possess both lipase and phospholipase activities. We suspected that aleurone beta-amylase may serve as a lipase inhibitor to block phospholipase activity and delay the senescence of aleurone cells. To examine the hypothesis, we test whether maize beta-amylase can serve as a lipase inhibitor.
In order to conduct the interaction assay of beta-amylase and lipase, we adopted a preparative electrophoresis method with a Prep Cell to purify beta-amylase proteins from germinating maize endosperms and soybean cotyledons. We pre-mixed tested proteins with lipid substrate prior to the addition of lipase preparation and assayed the lipid hydrolytic activity. Although maize beta-amylase did not affect the activity of partially purified maize lipase, both soybean and maize beta-amylases showed the inhibitory effect on the enzyme activity of pancreatic lipase. For testing if there were any physical interactions between lipase and beta-amylase molecules, we pre-incubated lipase with beta-amylase before adding substrate to the reaction mixture. No inhibitory effects of beta-amylases on either pancreatic or maize lipases could be detected. The data suggested that the inhibitory effect of beta-amylase on lipase is not due to the direct interaction of these two protein molecules.
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