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.
Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that 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.
Frederiks
Wilma M
WM
Department of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. w.m.frederiks@amc.uva.nl
van Marle
Jan
J
van Oven
Carel
C
Comin-Anduix
Begonya
B
Cascante
Marta
M
D000818:Animals; D002453:Cell Cycle; D045744:Cell Line, Tumor; D003470:Culture Media; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D010084:Oxidation-Reduction; D051381:Rats; D013778:Tetrazolium Salts
D003470:Culture Media; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; C075453:5-cyano-2,3-ditolyltetrazolium; D005954:Glucosephosphate Dehydrogenase