| Summary: | 碩士 === 國立臺灣大學 === 農業化學研究所 === 89 === Abstract
Sucrose-6-phosphate phosphatase (SPP, EC. 3.1.3.24) catalyzes the final step in the pathway of sucrose biosynthesis. The enzyme was purified from crude extract of leaves of sweet potato (Ipomoea batatas (L.) Lam) by DEAE-sephacel column, ammonium sulfate fractionation, Sephacryl S-200 and preparative electrophoresis. The partially purified SPP with 164- fold purification and specific activity of 11.5 μmol / min / mg protein was obtained. The molecular mass of the native sweet potato leaves SPP was estimated to be about 120 kD using gel filtration column. The SPP protein band shown in native PAGE by activity staining migrated between markers of 140 and 67 kD. The subunit mass was determined to be 50 kD by SDS-PAGE.
The enzyme is highly specific to sucrose-6F-phospate with a Km of 0.38 mM. The value is higher than its counterparts in rice leaves and spinach leaves, with Km of 0.065 mM and 0.105 mM, respectively. In contracst, Km of SPP from sweet potato leaves is lower than that from sweet potato tuberous root (0.59 mM). The stable enzyme activity is dependent on Mg2+ but inhibited by EDTA and divalent cations like Mn2+, Co2+, Zn2+ and Ca2+ in a concentration of 1 mM. The optimum temperature for SPP activity was between 40℃ and 50℃. The activity was optimal at pH 6.0 to 8.0. Sucrose is a weak inhibitor for SPP around 200 mM. SPS inhibited the hydrolysis of exogenous sucrose-6F-phospate by SPP when SPS was added in the absence of its substrate (i.e. UDP-Glc). But it stimulated SPP activity over 60% when the SPS substrates were present and used to generate sucrose-6F-phospate directly in the reaction. It is possible that SPS and SPP present as a complex in vivo for efficient biosynthesis of sucrose.
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