MEASUREMENT OF SUBCELLULAR METABOLITE LEVELS IN LEAVES BY FRACTIONATION OF FREEZE-STOPPED MATERIAL IN NONAQUEOUS MEDIA
MEASUREMENT OF SUBCELLULAR METABOLITE LEVELS IN LEAVES BY FRACTIONATION OF FREEZE-STOPPED MATERIAL IN NONAQUEOUS MEDIA
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DOI：
10.1104/pp.75.3.542
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发表时间：
1984-01-01
期刊：
影响因子：
7.4
通讯作者：
HELDT, HW
中科院分区：
文献类型：
--
作者：
GERHARDT, R;HELDT, HW
本文介绍了一种测定菠菜叶片叶绿体基质、细胞质和液泡中代谢产物含量的方法。菠菜叶子被冷冻，冷冻后的组织被碾碎并冻干。干燥后的物料在四氯化碳和庚烷混合物中超声均质，密度梯度离心分馏。对不同亚细胞区室中标记酶活性的测量表明，叶绿体物质主要出现在最轻的部分，细胞质物质出现在梯度的中间，而大多数液泡物质出现在最重的部分。利用测量的代谢物和标记酶在梯度各部分的分布，可以计算代谢物的亚细胞分布。作为第一个应用，新的分离技术被用于研究菠菜叶中苹果酸盐和蔗糖的亚细胞含量。结果表明，蔗糖和苹果酸盐的日变化显著，这两种物质主要位于液泡室。苹果酸盐在一天结束时的含量是晚上结束时的3倍。液泡中的蔗糖含量从一天结束时最大的45毫米下降到晚上结束时几乎无法检测到的约1毫米。
This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol and the vacuole of spinach (Spinacia oleracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane and fractionated by density gradient centrifugation. Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated. As a first application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About 3 times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole falls from a maximum of 45 mM at the end of the day to an almost undetectable value of approximately 1 mM at the end of the night.