Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements.
BACKGROUND
The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed.


METHODS
Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions.


RESULTS
Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves.


CONCLUSIONS
(1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.
Deutsch
M
M
The Jerome Schottenstein Cellscan Center for Early Detection of Cancer, Physics Department, Bar-Ilan University, Ramat-Gan 52900 Israel. arnon-sh@actcom.net.il
Kaufman
M
M
Shapiro
H
H
Zurgil
N
N
D001667:Binding, Competitive; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D007700:Kinetics; D020298:U937 Cells
D004798:Enzymes; D005452:Fluoresceins; C018506:diacetylfluorescein