【作者】 费美娟；

【导师】 王晓云；

【作者基本信息】 山东农业大学 ， 生物化学及分子生物学， 2004， 硕士

【摘要】 以小麦品种 pH82-2-2 为材料,研究了小麦叶片磷酸酯酶和类囊体膜磷酸酯酶的基本酶学性质?热稳定性?激活作用及抑制动力学?以期通过调节酶活力来调节光合速率以及为改善小麦磷素营养提供理论依据?主要研究结果如下: 1. 叶片磷酸酯酶水解 pNPP 的 Km值为 6.554 mmol/L?该酶的最适 pH为 6.8,在 pH 6.4~7.5 的范围内酶催化 pNPP 水解的活力较高;保温 10 min时的最适温度为 45℃,在 50℃,60℃,70℃保温时,该酶活力丧失 50%所需的时间分别为 12 min?3.75 min?1.8 min? 2. 不同的金属离子及络合剂对叶片磷酸酯酶活力有影响,1~5mmol/L 的 Mg2+和 Ca2+对该酶有激活作用,0.1~0.5 mmol/L 的 Zn2+?Cu2+和 Mn2+对酶活力则有不同程度的抑制作用?甲醇?乙醇和乙二醇对酶活力有抑制作用?氟化钠和磷酸氢二钠对该酶活力有抑制作用,其抑制类型分别为非竞争性和竞争性抑制,抑制常数分别为 1.076 mmol/L 和 2.47mmol/L? 3. 类囊体膜磷酸酯酶水解 pNPP 的 Km值为 3.914 mmol/L?该酶的最适 pH 为 4.6,保温 5 min 时的最适温度为 55℃,在 60℃,70℃,80℃保温时,该酶活力丧失 50%所需的时间分别为 4.5 min?2.8 min?2 min? 4. Mg2+,Ca2+和 Fe2+能够激活类囊体膜磷酸酯酶活力,Mn2+和 Cu2+则抑制酶活力?有机溶剂甲醇,乙醇和乙二醇对类囊体膜磷酸酯酶活力有 1<WP=6>费美娟:小麦叶片磷酸酯酶和类囊体膜磷酸酯酶生化性质及抑制动力学研究激活作用,其中乙醇的激活程度最强,其激活类型属于反竞争型?钼酸铵?偏钒酸铵?磷酸氢二钠和氟化钠都能抑制类囊体膜磷酸酯酶活力,抑制类型依次为反竞争性?非竞争性?竞争性和混合型?5. 初步研究表明,光/暗处理能够调节小麦类囊体膜磷酸酯酶的活力,光下处理的小麦类囊体膜磷酸酯酶活力总是高于暗处理下的酶活力;将小麦由光下转入暗中和由暗中转入光下时类囊体膜磷酸酯酶活力变化趋势分别呈"V"和"W"型?6. 小麦叶片磷酸酯酶和类囊体膜磷酸酯酶在生化性质方面具有一些共同点?在 pH 试验中它们除了最适 pH 以外都还有另外一个小峰,它们都可被 Mg2+和 Ca2+激活以及被钒酸盐和钼酸盐抑制?由这些结果可以看出,在小麦叶片和类囊体膜中都可能存在不同形式的磷酸酯酶?更多还原

【Abstract】 The enzymatic properties, heat stability, activation and inhibition kineticsof wheat leaf phosphatase and thylakoid membrane phosphatase were studiedusing wheat pH82-2-2, anticipating to supply theoretical base for regulatingphotosynthesis, improving phosphorus nutrition by regulating enzyme activity.The results were as follows: 1. The Km value for pNPP of leaf phosphatase was 6.554 mmol/L. Thephosphatase had a pH optimum of 6.8 and a temperature optimum of 45℃incubated for 10 min. The half-life of phosphatase at 50℃, 60℃ and 70℃were 12 min, 3.75 min and 1.8 min, respectively. 2. 1-5 mmol/LMg2+and Ca2+had effects of activation on the phosphataseand 0.1-0.5 mmol/L Zn2+, Cu2+ and Mn2+ had effects of inhibition. Methanol,ethanol and glycol inhibited the activity of leaf phosphatase. Fluoride andphosphate also inhibited the activity of leaf phosphatase and the inhibitiontype was noncompetitive and competitive according to Lineweaver-Burk plot,and the inhibition constants were 1.076 mmol/L and 2.47 mmol/L respectively. 3<WP=8>费美娟:小麦叶片磷酸酯酶和类囊体膜磷酸酯酶生化性质及抑制动力学研究 3. The thylakoid membrane phosphatase had a Km value for pNPP of3.914 mmol/L. It had a pH optimum of 4.6 belonging to acid phosphatase anda temperature optimum of 55℃. The half-life of phosphatase at 60℃, 70℃and 80℃ were 4.5 min, 2.8 min and 2 min respectively. The activity was lostcompletely when incubated at 80℃ for 9 min. 4. The thylakoid membrane phosphatase activity was activated by Mg2+,Ca2+and Fe2+, inhibited by Mn2+ and Cu2+. Methanol, ethanol and glycol allcould activate the activity, the activation degree of ethanol was the strongestand its activation type was uncompetitive. The activity of thylakoid membranephosphatase was inhibited by molybdate, vanadate, phosphate and fluoride,the inhibition type of them were uncompetitive, noncompetitive, competitiveand mixed, respectively. 5. The treatment of light/dark can regulate the activity of wheat thylakoidmembrane phosphatase, the activity of thylakoid membrane phosphatase underlight was higher than that of under darkness; the change trend of the activity ofwheat thylakoid membrane phosphatase were "V"and "W"form when treatedfrom light to darkness and from darkness to light. 6. The wheat leaf phosphatase and thylakoid membrane phosphatase hadsome common biochemical properties. In addition to the main pH activitypeak, there was a smaller peak; the activities were activated by Mg2+and Ca2+,and inhibited by molybdate and vanadate. All these results indicated that theremay be multiple phosphatases in wheat leaf and thylakoid membrane.更多还原