Studies on the soluble acid invertase and its inhibitor from sweet potato leaves

• Main Authors: Yung-Liang Wang, 王永樑
• Other Authors: Hsien-Yi Sung
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/18924254642210272848

博士 === 國立臺灣大學 === 農業化學研究所 === 91 === Invertases (b-fructofuranosidase; EC 3.2.1.26) are present in most plant organs in multiple forms, which are classified by pH optima (acid and neutral/alkaline), solubility properties (soluble and insoluble), and cellular locations. In various plants, there have been many reports on detection and characterization of invertase, but there is little information on the regulation of these enzymes. So far, there are some evidences about the regulation of invertase, such as light, environmental stimuli (wounding and pathogenic infection) and the reaction products, glucose and fructose. While the presence of proteinaceous inhibitor is provided another mechanism of modulated invertase activity. There are two soluble acid invertases (IbbFRUCT1 and IbbFRUCT2) being isolated from the leaves of sweet potato (Ipomoea batatas (L.) Lam). IbbFRUCT2 could be purified to apparent homogeneity by consecutive steps of ammonium sulfate fractionation, DEAE-Sephacel and Con A-Sepharose chromatographies. The molecular mass of the enzyme was 60 kD. Immunological analysis revealed that the two polypeptides, 40 kD and 30 kD polypeptides, which were found on SDS-PAGE during storage, were the degraded products of the 60 kD polypeptide. There were no similarities in the N-terminal amino acid sequences among these three polypeptides, whereas, amino acid sequence alignment data exhibits homologies of 69%, 40% and 90%, when the cDNA-derived amino acid sequence obtained from sweet potato leaves was compared. According to the amino acid alignment data, the soluble acid invertase degradation features from sweet potato leaves are: two cleavages steps produce the degraded products, with the first proteolytic product step (40 kD polypeptide) possibly the transition state, while, the 30 kD polypeptide might be ‘finished’ by the cleavages. Besides, the protease activity was detected during the purification process and participated in the degradation process, but the physiological function is still not known. A proteinaceous invertase inhibitor (ITI-L) was purified to electrophoretic homogeneity through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of ITI-L was 10 kD, with the characteristic of acid and heat stable. A Full-length cDNA encoded invertase inhibitor was obtained by a combination of RT-PCR, PCR and 5''''- and 3'''' RACE RT-PCR, starting with primers based on conserved amino acid sequences. The recombinant inhibitor was also obtained by means of in vitro translation system and the Ki value was measured in the presence of various concentrations of IN-L, as expected for simple linear competitive inhibition.