A comparative kinetic study on the conversion of fluoresceindiacetate to fluorescein in living cells and in vitro |
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Authors: | W Sontag |
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Institution: | (1) Kernforschungszentrum Karlsruhe, Institut für Strahlenbiologie, Postfach 3640, D-7500 Karlsruhe 1, Federal Republic of Germany |
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Abstract: | Summary 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 100–600 were derived. In addition, the action of chemical inhibitors and of elevated temperatures on the individual reaction steps was investigated. |
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