A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR INORGANIC-PHOSPHATE AND FOR MEASURING PHOSPHATE RELEASE KINETICS IN BIOLOGICAL-SYSTEMS
A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR INORGANIC-PHOSPHATE AND FOR MEASURING PHOSPHATE RELEASE KINETICS IN BIOLOGICAL-SYSTEMS
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DOI：
10.1073/pnas.89.11.4884
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发表时间：
1992-06-01
影响因子：
11.1
通讯作者：
WEBB, MR
中科院分区：
文献类型：
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作者：
WEBB, MR
本文提出了一种用2-氨基-6-巯基-7-甲基嘌呤核苷和嘌呤核苷磷酸化酶（嘌呤核苷：正磷酸核糖基转移酶，EC 2.4.2.1）测定无机磷（P（i））的分光光度法。该底物在pH 6.5-8.5下磷酸解时在360 nm处的吸光度增加，在pH 7.6下消光系数的变化为11，000 M-1.cm-1。两种底物与酶的米氏常数对于核苷为70 μ M，对于P（i）为26 μ M; k（cat）为40 s-1（25 ℃）。该测定法显示在至少低至2 μ M的浓度下定量溶液中的P（i）。它可用于测量P（i）从磷酸酶（如GTP酶和ATP酶）释放的动力学，通过耦合两种酶促反应。三个试验系统显示了该测定的实用性：甘油激酶+D-甘油醛作为ATP酶和肌动蛋白激活的肌球蛋白ATP酶，以及肌球蛋白亚片段1，水解ATP的单一周转，以与稳态ATP酶活性相同的速率常数释放P（i）。
A spectrophotometric method for the measurement of inorganic phosphate (P(i)) has been developed by using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This substrate gives an absorbance increase at 360 nm on phosphorolysis at pH 6.5-8.5, and at pH 7.6 the change in extinction coefficient is 11,000 M-1.cm-1. The Michaelis-Menten constants of the two substrates with the enzyme are 70-mu-M for the nucleoside and 26-mu-M for P(i); the k(cat) is 40 s-1 (25-degrees-C). The assay was shown to quantitate P(i) in solution at concentrations at least down to 2-mu-M. It can be used to measure the kinetics of P(i) release from phosphatases, such as GTPases and ATPases, by coupling the two enzymic reactions. The utility of this assay was shown by three test systems: glycerol kinase plus D-glyceraldehyde acting as an ATPase and actin-activated myosin ATPase, and myosin subfragment 1, hydrolyzing a single turnover of ATP, releasing P(i) with a rate constant the same as the steady-state ATPase activity.