Purification and characterization of type H starch phosphorylase from sweet potato

• Main Authors: Chen, Jia Yu, 陳家裕
• Other Authors: Zhuang, Rong Hui
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/22348264557848813604

碩士 === 國立臺灣大學 === 農業化學研究所 === 83 === Two types of starch phosphorylase (SP) were found in sweet potato roots according to their affinity to starch : a low-affinity L-SPand a high-affinity H-SP. L-SP was puried and studied extensively in its biochemical properties and molecular structure. Instead HSP was more difficult to achieve a convenient purification procedure and was not well characterized. Both types of SP were believed to catalyze the phosphorolysis of starch in plant cell,but solid evidence was still lacking to elucidate the real physiological role of this enzyme. In this study, H-SP was purified to apparant homogeneity, antibody against H-SP was prepared, and some biochemical properties were characterized. It was found that H-SP contained two 90 kD subunits, lacking the unique 78-amino acid insertion of L-SP. The molecule contained carbohydrate which was revealed on an electrophoretic gel stained specifically for sugar moiety. When the enzyme was visualized by activity staining a minor band appeared besides the major band. So isoforms of H-SP were suggested. The isoelectric point measured by isoelectric focusing was around 5.0. H-SP cross-reacted with the conventional antiserum raised against L-SP, and vice versa. But two monoclonal antibodies (J3b and H7c) against L-SP had no reaction with H-SP. J3b and H7c were reported to bind to the eptiopes located on the L78 of L-SP. Kinetic studies showed that H-SP preferred glycogen as its substrate than starch, and starch was the preference for L-SP.