Examples Of Metabolic Determinations

Redox state and influence of cell milieu on enzymatic activities

The thiole glutathione (GSH) is the most important nonenzymatic antioxidant in organisms and exists in considerably high concentrations in all cell types. GSH can react with electrophilic compounds or free radicals. The content of GSH in aging tissues generally declines, and it is assumed that this decline reduces the capacity to

Larval Pupal- Adult- stage

Larval Pupal- Adult- stage

400 ^ 350 300 250 200 150 100

Figure 21.9. Glutathione content of Phormia flies in different life stages.

defend toxic effects of free radicals. Buthionin sulfoximine (BSO) is an irreversible inhibitor of the y-glutamylcystein synthetase, which is a key enzyme for the synthesis of GSH. BSO can be used to empty the intracellular stores of GSH. This allows conclusions about the protective role of GSH.

In Phormia there is a strong correlation between life stage and the GSH content (Figure 21.9).

Treatment with BSO for 11 days reduces the GSH content to 10% compared with the control. After this treatment, a resynthesis of GSH is observed only in younger flies.

Similarly, Phormia can be used to study the age-dependent capability of xenobiotics via the GSH-S-transferase (GST) system for detoxification (Collatz and Flury, 1992). Using the analgetic drug Paracetamol, we were able to show that both a reduced GSH level and a diminished activity of GST lead to an increasing mortality of old flies after drug injection.

It is a common phenomenon that the redox state of an old organism is shifted towards the oxidized site. Phormia is no exception. Together with the reduced GSH content, the quotient of the NAD/NADH and NADP/NADPH redox system is responsible for the redox state of the cell (Figure 21.10).

It is important to note that a change of the redox state, and with this the cell milieu, can alter enzyme activities. Jaekel in our laboratory showed this for purified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from young and old flies (Figure 21.11).

The kinetic properties of "young" and "old" enzymes are determined by the age-appropriate in vivo concentrations of NAD and NADH. "Old" enzyme reacts under "young" physiological conditions with an increase of Vmax and Km. In contrast "young" enzyme reacts under "old" physiological conditions with a reduction of Vmax and Km.

NAD/NADH quotient

NAD/NADH quotient

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