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Activity expression of L-myo-inositol-1-phosphate synthase in suspension-cultured rice cells

悬浮培养水稻细胞中L-肌醇-1-磷酸合酶的活性表达

基本信息

批准号：
63560077
负责人：
IGAUE Ikuo
金额：
$ 1.22万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

Activity expression, activity change, enzyme purification and properties of L-myo-inositol- 1-phosphate synthase(MIPS) were analyzed employing rice cells in suspension-culture. myo-Inositol(MI) biosynthesis and a role in controlling metabolism also were investigated. It was shown that in MI deficient medium at the early logarithmic stage, the activity of MIPS is prominent, but in medium to which MI was, is suppressed. Sigmoid curve of the enzyme activity with critical point, 20 mg MI/1, was observed. Furthermore, the activity change at MI concentration was examined, Km was unchanged but Vmax varied in proportion to the activity. It was, therefore, suggested that the activity regulation by MI was not inhibition of enzyme activity but synthetic regulation of enzyme molecules.Using 20 mM Tris-HC1 buffer(pH 7.5) containing 20%(V/V) glycerin, 2 mM 2-mercaptoethanol, the purification of enzyme was performed at 3ﾟC. The enzyme was isolated from the cell extract and purified 600-fold with a 6% yield by ammonium sulfate fractional precipitation, and chromatographies on DEAE-Toyopearl, butyl-Toyopearl, Hydroxylapatite, Cellulofine GCL-2000 and chromatofocusing Attempts to obtain the homogeneous enzyme were unsuccessful.On the other hand, due to the presence of the powerful non-specific acid phosphatase in the enzyme, periodate oxidation-malachite green method was adopted instead of bioassay method, and subsequent assay was simplified. NH^+_ accelerates the rate of reaction 4-fold. The enzyme showed pH optimum at 8.0 with Km value of 0.79 mM. The activity was about 15 - 16% NAD^+-independent. The activity was remarkably inhibited by a number of pentose phosphate pathway: 6-phosphogluconate, riburose-5-phosphate, ribose-5-phosphate and fructose-6-phospha-Le. The enzyme activity was inhibited by Ag^+, Cu^<2+>, Zn^<2+>, Ba^<2+> and Fe^<3+>. Some sulfhydryl reagent and nucleotide-coenzyme used also were inhibitory.

以水稻细胞悬浮培养为材料，研究了L-肌醇-1-磷酸合成酶（MIPS）的活性表达、活性变化、酶的纯化及性质。还研究了肌醇（MI）的生物合成和在控制代谢中的作用。结果表明，在对数生长初期，MI缺乏的培养基中，MIPS的活性显著，而在MI达到的培养基中，MIPS的活性受到抑制。观察到酶活性与临界点20 mg MI/l的S形曲线。此外，在MI浓度下检查活性变化，Km不变，但Vmax与活性成比例变化。用含20%（V/V）甘油、2 mM 2-巯基乙醇的20 mMTris-HCl缓冲液（pH7.5），在3 ℃下进行酶的纯化。从细胞提取物中分离出该酶，并通过硫酸铵分级沉淀以6%的产率纯化600倍，并在DEAE-Toyopropylene、butyl-Toyopropylene、Hydroxyapatite、Cellulofine GCL-2000和色谱聚焦上进行层析，试图获得均相酶，但没有成功。采用高碘酸盐氧化-孔雀石绿绿色法代替生物测定法，简化了后续测定。NH^+_使反应速率加快4倍。该酶的最适pH为8.0，Km值为0.79 mM，酶活力与NAD^+的依赖性为15 - 16%。该酶活性可被6-磷酸葡萄糖酸、核酮糖-5-磷酸、核糖-5-磷酸和果糖-6-磷酸等戊糖磷酸途径显著抑制。Ag^+、Cu^<2+>、Zn^<2 +>、Ba^<2+>和Fe^<3+>对酶活性有抑制作用。使用的一些巯基试剂和核苷酸-辅酶也有抑制作用。