Measurement of Enzyme Activities in Living Cells

描述（由申请人提供）：用于表征细胞和 细胞的亚细胞化学异质性将在 确定疾病发作与化学环境之间的关系 细胞存在的地方。生物学中大多数酶活性的测定 系统已经在大量均质化细胞上进行， 不能辨别细胞间异质性，提供了差的时间 分解，并破坏酶在其中的天然胞质环境。 功能在少数情况下，研究人员已经改进了这种传统的 方法通过开发用于测量总酶活性的方法 单细胞，但这些技术还没有能力检查 在亚细胞水平上的活动变化或用于跟踪时间演变 一个神经元。 在这些研究中，我们将开发一种全新的测量策略， 在活神经元的亚细胞水平上的酶活性。光谱学上 和特定蛋白激酶的酶促“笼状”底物（即，一 磷酸化酶）将被引入培养细胞的胞质溶胶中， 并将在一个微米长的轴突内被光解激活， 树突状区域在一个明确定义的时间点后，电和/或 化学刺激经过一段短暂的潜伏期， 催化底物磷酸化，将施加电场 纵向到轴突/树突，引起（细胞内）磷酸化 将产物从未反应的底物中分离出来。的 特异性激酶的活性将使用高灵敏度 荧光显微镜来测量两种材料的相对量 电泳条带重要的是，感兴趣的细胞不是有目的地 在这些测量中被破坏，打开了多个 在同一个神经元上进行测量。这些研究涉及设计， 一种新技术--“轴突毛细血管”的实施和评价 电泳”-这将打开重要的窗口，进入亚细胞 神经元化学这种非假设驱动的工作代表了一个强有力的匹配 NIMH探索/发展（R21）资助计划的目标 （PA-OO-073）。

DESCRIPTION (provided by applicant): Techniques for characterizing cellular and subcellular chemical heterogeneities of cells will play a vital role in defining the relationships between disease onset and the chemical environments in which cells exist. Most determinations of enzyme activities in biological systems have been performed on large numbers of homogenized cells, an approach that cannot discern cell-to-cell heterogeneities, provides poor temporal resolution, and destroys the native cytosolic environments in which enzymes function. In a few instances, researchers have improved on this conventional approach by developing methods for measuring the total enzyme activity of single cells, but these techniques have not had capabilities for examining variations in activity at subcellular levels or for tracking the time-evolution of an individual neuron. In these studies, we will develop a fundamentally new strategy for measuring enzyme activities at the subcellular level in live neurons. A spectroscopically and enzymatically "caged" substrate for a specific protein kinase (i.e., a phosphorylating enzyme) will be introduced to the cytosol of a cultured cell, and will be photolytically activated within a micrometer-long axonal or dendritic region at a well-defined time point following an electrical and/or chemical stimulus. After a brief incubation period in which the enzyme can catalyze substrate phosphorylation, an electric field will be applied longitudinally to the axon/dendrite, causing (intracellular) phosphorylated product to electrophoretically fractionate from unreacted substrate. The activity of the specific kinase will be determined using a high-sensitivity fluorescence microscope to measure the relative amounts of material in the two electrophoretic bands. Importantly, the cell-of-interest is not purposefully disrupted in these measurements, opening the possibility for multiple measurements on the same neuron. These studies involve the design, implementation, and evaluation of a new technology - "axonal capillary electrophoresis" - that will open important windows into the subcellular chemistry of neurons. This non-hypothesis-driven work represents a strong match to the objectives of the NIMH Exploratory/Developmental (R21) Grant Program (PA-OO-073).