Rapid measurement of low levels of sodium, potassium-ATPase activity by ascorbic acid reduction without strong acid.
Rapid measurement of low levels of sodium, potassium-ATPase activity by ascorbic acid reduction without strong acid.
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无需强酸即可通过抗坏血酸还原快速测量低水平的钠、钾-ATP 酶活性。
DOI：
10.1006/abio.2001.5407
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Spur,BW
中科院分区：
文献类型：
--
作者：
Spokas,EG;Spur,BW
考虑Eqs. [1]和[2]说明了可逆抑制剂（I）保护不被不可逆抑制剂标记的瞬时性质，这是FSBA保护方法的主要责任。例如，如果将FSBA的浓度固定在0.1 Kb（其可逆结合常数）和klab 1 min-1，则在10 min反应中标记83%的酶。包括浓度为其KI四倍的可逆抑制剂仅将标记的激酶的量减少至32%。从图1中可以清楚地看出实验含义：需要毫摩尔量的ATP来阻断FSBA的不可逆标记。因此，在BSA和ERK 2的标记过程中缺乏可观察到的5 mM ATP竞争可能是由于对ATP的亲和力低或标记在ATP结合位点之外;图1中的数据不允许进行区分。因此，在有限的条件下，将通过实验观察到对标记的保护;即，必须标记足够的酶以检测，但不能太多，保护将无法检测到。在解释来自标记保护实验的数据时，必须考虑可逆抑制剂提供的瞬时保护和蛋白激酶标记反应的动力学异质性。尽管如此，研究人员在意识到这些局限性后，可以使用FSBA标记的保护来评估纳摩尔浓度下蛋白激酶混合物的抑制模式和抑制剂特异性，正如我们在这里报道的那样。我们感谢默克研究实验室的Richard Cummings博士和Alice Marcy博士对手稿的严格审查。
Consideration of Eqs.[1] and [2] illustrate the transient nature of protection by a reversible inhibitor (I) from labeling by an irreversible one, which is the principal liability of the FSBA protection method. For example, if the concentration of FSBA is fixed at 0.1 Kb (its reversible binding constant) and klab 1 min 1 then 83% of the enzyme is labeled in a 10-min reaction. Including a reversible inhibitor at a concentration fourfold its KI only reduces the amount of labeled kinase to 32%. The experimental implications are clear from Fig. 1: millimolar amounts of ATP are required to block the irreversible labeling by FSBA. Thus the lack of observable competition by 5 mM ATP during labeling of BSA and ERK2 may owe to low affinity for ATP or to labeling outside of the ATP binding site; the data in Fig. 1 do not allow a distinction to be made. Hence there are limited conditions under which protection from labeling will be observed experimentally; ie, one must label enough enzyme to detect but not so much that protection will be undetectable. The transient protection provided by reversible inhibitors and the kinetic heterogeneity of protein kinase labeling reactions must be considered when interpreting data from labeling protection experiments. Nonetheless, researchers armed with an awareness of these limitations can use protection from FSBA labeling to assess mode of inhibition and inhibitor specificity in mixtures of protein kinases at nanomolar concentrations, as we have reported here.Acknowledgments. We thank Dr. Richard Cummings and Dr. Alice Marcy of Merck Research Laboratories for critical review of the manuscript.