Determination of individual cell Michaelis-Menten constants

Merav Sunray, Naomi Zurgil, Yana Shafran, Mordechai Deutsch

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)8-16
Number of pages9
JournalCytometry
Volume47
Issue number1
DOIs
StatePublished - 1 Jan 2002

Bibliographical note

Publisher Copyright:
Copyright 2001 Wiley-Liss, Inc.

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