Background Numerous studies have shown that the intracellular redox potential of
ID: 274979 • Letter: B
Question
Background
Numerous studies have shown that the intracellular redox potential of the cell is important to cell growth. Cellular redox potential can be determined by the amounts of the reduced coenzyme NADPH, a principal product of the oxidative branch of the pentose phosphate pathway (PPP). The investigators in the study presented here sought to demonstrate links between the activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) activity, cellular NADPH concentrations, and rates of cell growth. Previous studies have shown that the glucose-6- phosphate dehydrogenase enzyme can be activated on the order of minutes or even seconds, possibly through the action of growth factors that release a bound, inactive G6PD to the cytosol, where, via a mechanism that might involve tyrosine phosphorylation of a membrane-bound receptor, the unbound G6PD translocates to the cytosol and becomes active.
NADPH is important to the cell in a variety of ways. The reduced coenzyme can react with potential damaging oxidizing agents, ridding the cell of these agents before they can damage important cellular components.
Question
In the first experiment reported in this study, the authors stimulated cell growth through a variety of means; then measured corresponding G6PD activity of cultured fibroblasts. (Cells grown in culture are typically grown in a medium containing 10% fetal calf serum, a medium rich in growth factors.) The investigators grew fibroblasts in the absence of serum (serum- starved) to serve as a control. Cells were then treated with 10% fetal calf serum followed by the serum-starvation treatment; other cells were not starved. The results are shown in Figure 32.2. After the enzyme activity was measured, the cells were lysed and analyzed by Western blotting using a G6PD antibody. The results are of the Western blot are shown in Figure 32.3. What is your interpretation of these results? Be quantitative in your assessment of the data for both the activity assays and the Western blot. Why was the enzyme activity of PGD (6-phosphogluconate dehydrogenase) also investigated under the same conditions?
100 E 80 OG6PD PGD 2 60 40 20 2 Serum starved yes P lus Serum yes yes no no yes Figure 32.2: Fibroblasts were (1) serum-starved for 48 hours (2) serum-starved for 48 hours and then stimulated with 10% serum, and (3) actively grown in medium containing 10% fetal calf serum. Enzyme activity was measured in lysates containing the same number of cells (from Tian, et al, 1998).Explanation / Answer
the experiments were done to analyze G6PD in growing fibroblast cells. Three treatment options were taken-1. cells with serum starved and without growth factor, 2) cells which were serum starved and then 10% FCS were added as growth factor and 3) cells which were grown on normal growth medium containg 10% FCS. If we see the result, when cells were activated by the applicagtion of serum, the amount of G6PD also increased. Activity of G6PD get increased alomst 2 fold when serume is added to the starvaed cell ( condition 1 to condition 2): whereas, when cells are grown on normal medium which contains 10% serum, the G6PD level was almost 4 fold high in comparison to serum starved cells( condition 1 to condition 3). Thus its clear from the experiment that serum increased the activity of G6PD.
Now, they also checked the protein level of G6PD to confirm the increased activity of G6PD was due to the presence of incresaed amount of G6PD protein. Its shown in the western blot, the presence of serum increased the level of G6PD proein level.
As NADPH produced by both the enzymes G6PD and PGD in the pentose phosphate pathway, researcher wanted to check both the enzyme level in the experimental condition. However, PGD level was almost not affected by the starvation and addition of growth factors.
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