常见水生蕨类植物成熟孢子果中的葡萄糖生成果糖-1,6-双磷酸酶:细细细马齿藻(Polypodiopsida)中该酶的部分纯化和基本性质

S. Ghosh, M. Das, S. Basu, J. Adhikari
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引用次数: 0

摘要

本通讯报道了糖异生果糖-1,6-二磷酸酶(FBPase;EC 3.1.3.11)在三种常见的异孢子水生蕨类植物(Marsilea minuta, Salvinia natans和Azolla pinnata)中进行了检测,并描述了从Marsilea minuta成熟孢子中部分纯化其的方法。从马silea minuta, Salvinia natans和Azolla pinnata中获得的胞质FBPase被认为是糖异生酶,因为它在外部给药的葡萄糖存在下剧烈的分解代谢失活,并且对光合光照明不敏感。通过低速离心(11,400 × g), 30-80%硫酸铵分离,然后使用CM-Cellulose, Sephadex g -200和Ultrogel AcA 34等基质进行层析,从Marsilea minuta成熟孢子实中部分纯化出细胞内糖异生FBPase,比匀浆高22倍。记录了部分纯化的FBPase在非变性条件下在PAGE中的分布。酶活性随蛋白质浓度线性增加至约100µg,随时间增加至75分钟。最适温度为35℃。采用d -果糖-1,6-二磷酸(D-FBP,底物)在0.0 ~ 1.0 mM范围内,以0.1 mM的浓度间隔对FBPase进行了底物浓度的影响和动力学分析。利用Prism 8软件(Graph Pad)进行非线性回归动力学测定,FBPase D-FBP的Km值为0.06129 mM, Vmax为4525 nmol Pi释放(mg)-1蛋白h-1。该酶在7.0至8.0的狭窄pH范围内起作用,在pH 7.5时达到最大值。该酶受Mg2+的显著刺激,受Hg2+、Cu2+和Zn2+的强烈抑制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)
The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.
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来源期刊
CiteScore
0.80
自引率
0.00%
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36
审稿时长
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