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Summary form only given. A novel methodology for the measurement and analysis of apparent Km (Michaelis-Menten constant) and V/sub MAX/ values of individual cells is suggested. It is based on a mathematical model that considers substrate influx into the cell, its intracellular enzymatically hydrolysis, and the product efflux. The mathematical formulation was linearly approximated in order to analyze intracellular substrate conversion characteristics via Michaelis-Menten theory. Utilizing microsive component, a product of MEMS technology, in static cytometry, the time dependency of the Fluorescence Intensity (FI(t)) emitted from pre localized and defined fluorescein-diacetate (FDA) stained cells was recorded. This requires a high-level of periodical measurements of the same individual cells, which are sequentially exposed to various fluorogenic substrate concentrations. Model simulations were found to correlate with experimental results. Differences in distributions of individual Km and V/sub MAX/ values of cells incubated with and without Phytohaemagglutinin (PHA) were evident. Average Km and VMAX of PHA stimulated cells increased by 99% and 540% correspondingly. It is believed that this study may provide a tool for assessing intracellular enzymatic activity in individual intact cells under defined physiologic conditions. This may open new vistas in various areas, giving answers to critical questions arising in the fields of cell and developmental biology, immunology, oncology, and pharmacology. |
