Determination of individual cell Michaelis-Menten constants.
BACKGROUND
A novel methodology for the measurement and analysis of apparent K(M) (Michaelis-Menten constant) and V(MAX) values of individual cells is suggested. It is based on a mathematical model that considers substrate influx into the cell, its intracellular enzymatic hydrolysis, and the product efflux. The mathematical formulation was approximated linearly in order to analyze intracellular substrate conversion characteristics via Michaelis-Menten theory.


METHODS
Utilizing static cytometry, the time dependence of the fluorescence intensity [FI(t)] emitted from prelocalized and defined FDA stained cells was recorded. This required frequent periodical measurements of the same cells, which are sequentially exposed to various fluorogenic substrate concentrations.


RESULTS
Model simulations correlated with experimental results. Differences in distributions of individual K(M) and V(MAX) values of cells incubated with and without PHA were evident. Average K(M) and V(MAX) values of PHA-stimulated cells increased by 99% and 540%, respectively.


CONCLUSIONS
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 field of cell and developmental biology, immunology, oncology, and pharmacology.
Sunray
Merav
M
The Jerome Schottenstein Center for Early Detection of Cancer, Department of Physics, Bar-Ilan University, Ramat-Gan, Israel.
Zurgil
Naomi
N
Shafran
Yana
Y
Deutsch
Mordechai
M
D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008934:Mitogens / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D015203:Reproducibility of Results
D008934:Mitogens; D010835:Phytohemagglutinins