An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.
An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.
复制标题
葡萄糖 6-磷酸磷酸酶的阴离子交换放射测定：用于内质网囊泡的拓扑研究。
DOI：
10.1016/0003-2697(92)90374-g
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发表时间：
1992
影响因子：
2.9
通讯作者：
Waechter,CJ
中科院分区：
文献类型：
--
作者：
Rush,JS;Waechter,CJ
提出了用纯化的酵母己糖激酶和未标记的 ATP 酶法制备 [2-3H] 甘露糖 6-磷酸 (Man 6-P) 的简单程序。酶促合成的 [2-3H]Man 6-P 在新的葡萄糖 6-磷酸 (Glc 6-P) 磷酸酶快速测定中用作放射性标记底物。测定程序的原理是，未反应的底物 [2-3H]Man 6-P 由阴离子交换树脂 AG 1-X8（乙酸盐）保留，而酶产物 [2-3H] 甘露糖直接洗脱到闪烁计数瓶中。当通过新的色谱测定法测定与小鼠肝内质网 (ER) 囊泡相关的 Glc 6-P 磷酸酶活性时，观察到相同的特征潜伏期和特性，正如常用的无机磷酸盐比色测定法所测定的那样。所描述的阴离子交换放射测定应该可用于与来自肝脏和肾脏 ER 的膜囊泡相关的酶的各种拓扑研究。
A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.