| Summary: | 碩士 === 東吳大學 === 化學學系 === 86 === Phosphoenolpyruvate Carboxylase (EC 4.1.1.31) has been success-fully purified from rice roots by ammonium sulfate precipitation, and the series of chromatographic steps, inducing hydrophobic interaction, Gel filtration, and ion exchange chromatography to reach a final 110 purification fold and about 20 % of recovery yield. The native molecular mass was determined to be 418 kDa by gel filtration chromatography, whereas a molecular mass of 105 kDa was obtained by SDS-polyacrylamimde gel electrophoresis, indicating that the enzyme is a homotetramer. The PEPC activity depends upon the pH and shows a maximum activity at pH 8.2. PEPC was further characterized at the physiological pH 7.5. The results showed that KM for phosphoenol-pyruvate (PEP), HCO3-, and Mg2+ were 0.27, 0.28, and 1.26 mM, respectively. Similar to previous observation, either inhibition by malate or activation by glucose-6-phosphate or fructose-6-phosphate, PEPC activity showed a maximum repression or stimulation under physiological pH whereas only slightly effects under optimal pH conditions by malate or sugar phosphate were observed. Ki for malate were determined as 2.45 mM at pH 7.0, 29.54 mM at pH 7.5, and 59.1 mM at pH 8.0, respectively. On the other hand, Activation by G6P or F6P showed 136 % and 152 % at pH 7.0, 135 % and 140 % at pH 7.5, and 122 % and 120 % at pH 8.0, respectively. However, We have observed that PEPC activity was stimulated 126% when the malate concentration was under 5 mM at physiological pH. This result implied the importance of malate derived by PEPC in the root tissue.
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