Measurement of Enzyme Activities in Living Cells

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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).