A comparative kinetic study on the conversion of fluoresceindiacetate to fluorescein in living cells and in vitro.
The kinetics of fluoresceindiacetate (FDA)-hydrolysis in erythrocytes, CHO-cells and homogenized cells was investigated by means of fluorescence spectroscopy. At a 10(-6)m FDA concentration the enzymatic conversion in the homogenized cells can be described by first order reactions. When studying intact cells, total kinetics requires the addition of a first order transport term. Consequently, the total hydrolytic process can be described by three exponentials. The following model was set up on the basis of the data obtained: FDA, which exists in two sterically differing conformations, reaches the cell via an "active transport mechanism", where the two conformations are converted at different rates into fluorescein by an enzymatic reaction. Intracellular FDA-enrichment factors of 100-6-- were derived. In addition, the action of chemical inhibitors and of elevated temperatures on the individual reaction steps was investigated.
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D004912:Erythrocytes / Q000201:enzymology; D005452:Fluoresceins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D002265:Carboxylic Ester Hydrolases