*3.6. Hexokinase Activity*

The liver of arthritic rats presents a considerably increased glucose phosphorylation capacity [30], which is diminished when the rats are treated with MeJA [9]. For these reasons and because glucose oxidation may be related to the oxidative state of the brain tissue, experiments were done in which the influence of arthritis and MeJA on the hexokinase activity was investigated. Figure 6 illustrates the results that were obtained. Figure 6a shows the measurements of the hexokinase activity in the cytosolic fraction of brains from healthy and arthritic rats, treated or not with the current different MeJA doses. Arthritis had no significant effect on the hexokinase activity present in the cytosolic

fraction. The MeJA treatment, on the other hand, increased the hexokinase activity in the cytosolic fraction obtained from healthy rats. A similar effect was found in arthritic rats treated with MeJA, with a maximal effect at the dose of 300 mg/kg.

**Figure 6.** Effects of MeJA treatment on hexokinase activity. (**a**) Hexokinase activities in the cytosolic fraction of brains from healthy and arthritic rats, treated or not with different MeJA doses (C, controls; C300, controls treated with 300 mg/kg MeJA; A, arthritic rats; A75, A150 and A300, arthritic rats treated with 75, 150 and 300 mg/kg MeJA, respectively). (**b**) Hexokinase activities in the cytosolic fraction of brains from healthy and arthritic rats incubated with various MeJA concentrations with or without previous centrifugation to eliminate mitochondria and other cell components. Data are the means ± standard errors of the mean of five animals for each experimental condition. Statistical analysis: ANOVA one-way with Newman–Keuls post-hoc testing; \**p* < 0.05, different from the corresponding controls (C); #*p* < 0.05, different from non-treated arthritic rats (A).

In brain cells the hexokinase is attached to the mitochondrial membrane and it is known that MeJA is able to detach the enzyme [5]. Since mitochondria were absent in the cytosolic fraction used for the hexokinase assay, the possibility exists that the higher hexokinase activities found in the brain of MeJA-treated animals can actually represent detached enzyme due to the continuous presence of MeJA in the tissue. To investigate this possibility the series of experiments shown in Figure 6b were done. MeJA was added at varying concentrations to the low-speed centrifugation supernatant of the brain homogenate either before or after the high-speed centrifugation for precipitating the mitochondria and other cell components. The time of exposure to MeJA was in all cases 30 min. The hexokinase activity in the cytosolic fractions was represented against the MeJA concentrations. Up to the MeJA concentration of 2.5 mM, precipitation of the mitochondria did not modify the activity of hexokinase found in the cytosolic fraction, although a small increasing tendency was found for both types of incubations. Above this concentration, however, the hexokinase activity continued to increase with the MeJA concentration in those preparations in which the mitochondria were still present when the compound was added. In the preparations in which the mitochondria had been precipitated previous to the addition of MeJA, the hexokinase activity ceased to increase. The enhancement of the activity when the incubation containing mitochondria was used probably reflects the detachment of the hexokinase bound to the organelles caused by MeJA. The detached hexokinase, shown in Figure 6b, calculated as the difference between incubations with and without mitochondria, was similar for the healthy and arthritic condition.
