Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon.
Development and Characterization of Quantitative, High-Throughput-Compatible Assays for Proteolytic Degradation of Glucagon.
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DOI：
10.1177/2472555218786509
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发表时间：
2018-12
期刊：
影响因子：
--
通讯作者：
Leissring MA
中科院分区：
文献类型：
--
作者：
Suire CN;Lane S;Leissring MA
胰高血糖素是一种重要的肽类激素，参与空腹状态下血糖的调节。虽然胰高血糖素产生和分泌的基础过程是很好地理解的，但对其降解的了解要少得多，可以想象的是，为了治疗益处，可以操纵胰高血糖素的降解。我们在这里描述的开发新的测定胰高血糖素降解，基于修饰的胰高血糖素肽（FBG）标记的荧光素和生物素在N-和C-末端，分别。FBG内任何肽键处的蛋白水解将荧光标记与生物素标签分离，其可以多种方式定量。在一种不需要专门设备的方法中，使用中性抗生物素蛋白-琼脂糖珠将完整的FBG与切割的荧光片段分离，并通过荧光定量水解。在另一种高通量相容的方法中，加入未修饰的抗生物素蛋白后，使用荧光偏振定量水解程度。使用已知的胰高血糖素蛋白酶，我们确认FBG在与未修饰的胰高血糖素相似的位点被切割，并利用这两种方法来量化FBG降解的动力学参数。我们进一步表明，基于荧光偏振的测定在高通量、混合和测量格式中表现得非常好（Z '因子值>0.80）。
Glucagon is a vital peptide hormone involved in the regulation of blood sugar under fasting conditions. While the processes underlying glucagon production and secretion are well understood, far less is known about its degradation, which could conceivably be manipulated pharmacologically for therapeutic benefit. We describe here the development of novel assays for glucagon degradation, based on a modified glucagon peptide (FBG) labeled with fluorescein and biotin at the N- and C-termini, respectively. Proteolysis at any peptide bond within FBG separates the fluorescent label from the biotin tag, which can be quantified in multiple ways. In one method requiring no specialized equipment, intact FBG is separated from the cleaved fluoresceinated fragments using neutravidin-agarose beads, and hydrolysis is quantified by fluorescence. In an alternative, high-throughput-compatible method, the degree of hydrolysis is quantified using fluorescence polarization after addition of unmodified avidin. Using a known glucagon protease, we confirm that FBG is cleaved at similar sites as unmodified glucagon and utilize both methods to quantify the kinetic parameters of FBG degradation. We show further that the fluorescence polarization-based assay performs exceptionally well (Z’-factor values >0.80) in high-throughput, mix-and-measure format.
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影响因子：
3.7
作者：
Cabrol C;Huzarska MA;Dinolfo C;Rodriguez MC;Reinstatler L;Ni J;Yeh LA;Cuny GD;Stein RL;Selkoe DJ;Leissring MA
通讯作者：
Leissring MA
影响因子：
4.8
作者：
Leissring, MA;Lu, A;Selkoe, DJ
通讯作者：
Selkoe, DJ
DOI：
10.1073/pnas.0801261105
发表时间：
2008-07-15
影响因子：
11.1
作者：
Neant-Fery, Marie;Garcia-Ordonez, Ruben D.;Leissring, Malcolm A.
通讯作者：
Leissring, Malcolm A.
影响因子：
4.8
作者：
Hinke, SA;Pospisilik, JA;McIntosh, CHS
通讯作者：
McIntosh, CHS
影响因子：
7.3
作者：
Abdul-Hay, Samer O.;Lane, Amy L.;Leissring, Malcolm A.
通讯作者：
Leissring, Malcolm A.