Improved Localization of Glucose-6-Phosphate Dehydrogenase Activity in Cells with 5-Cyano-2,3-ditolyl-tetrazolium Chloride as Fluorescent Redox Dye Reveals Its Cell Cycle-dependent Regulation
Wilma M. Frederiks 1*, Jan van Marle 1, Carel van Oven 1, Begonya Comin-Anduix 1 and Marta Cascante 1
1 Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, Amsterdam, The Netherlands (WMF,JvM,CvO); Department of Surgery, University of California at Los Angeles, Los Angeles, California (BC-A); and Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain (MC)
* To whom correspondence should be addressed. E-mail: w.m.frederiks{at}amc.uva.nl.
Submitted on February 24, 2005
Accepted on 12 July 2005
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Abstract |
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Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt which gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 ± 4% and 43 ± 4% higher, respectively than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.
Key Words:
glucose-6-phosphate dehydrogenase, CTC, tetrazolium salt, confocal laser scanning microscopy, cell cycle
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