**2.1 Inorganic polyphosphate metabolism in tick mitochondria**

Mitochondria from tick embryos in the segmentation stage (ninth day after oviposition) were isolated and respiration was measured using pyruvate as the substrate. The rate of oxygen consumption was 30 nmol/min/mg protein, and the respiratory control ratio (RCR) was 6.5. The process was KCN- and oligomycin-sensitive, his fraction exhibited an ATP hydrolyses azide sensitivity, a mitochondrial marker higher than 80%, and no activity of glucose-6-phosphate dehydrogenase, a cytosol marker, was detected (Table 1).


Table 1. Mitochondrial characterization

Once the mitochondria were characterized, mitochondria in eggs in the segmentation stage (ninth day after ovoposition) were isolated and exopolyphosphatase activity was measured in order to evaluate the regulation of its activity. The influence of NADH, phosphate, and ADP was investigated in concentrations ranging from 0.1 to 2.0 mM. The activity of exopolyphosphatase was stimulated by a factor of two by NADH, whereas its activity was completely inhibited by 2 mM phosphate and slightly stimulated by ADP (Figure 5A). The activity of exopolyphosphatase was also measured during mitochondrial respiration using pyruvate as the substrate and polyphosphate as the only phosphate source. During this assay, the addition of a small amounts of ADP (0.2 mM) induced state 3 (phosphorylating respiratory rate) followed by state 4 (non-phosphorylating respiratory rate), when all of the ADP was converted to ATP.

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks 149

Thus, during state 3, a balance existed between the release of phosphate by exopolyphosphatase and ATP synthesis, since exopolyphosphatase activity was measured by the amount of phosphate present. The exopolyphosphatase activity increased during mitochondrial respiration when pyruvate and ADP were added. This increase did not occur without the addition of ADP, indicating that exopolyphosphatase is stimulated during state 3 and that the rate of phosphate release is higher than the rate of ATP synthesis. Indeed, the stimulatory effect was antagonized by olygomicin, an ATP synthase inhibitor (Figure 5B). These data suggest that mitochondrial exopolyphosphatase activity is regulated by

Furthermore, it was possible to measure ADP-dependent mitochondrial oxygen consumption in the presence of polyphosphate and in the absence of any other phosphate source. This oxygen consumption was observed using polyphosphate3 and polyphosphate15; however, the consumption was higher with polyphosphaste3. On the other hand, heparin, an exopolyphosphatase inhibitor, blocked oxygen consumption, which was recovered when 5 mM phosphate was added and was again interrupted by the addition of oligomycin, an ATP-synthase inhibitor (Figure 6). These results suggest that polyphosphate was used as a phosphate donor for ATP synthesis due to the mitochondrial coupling observed when mitochondrial respiration was interrupted by oligomycin and the existence of membrane exopolyphosphatase in this process, due to the inhibition by heparin, which cannot cross the mitochondrial membrane and has its active site oriented toward the external face of the membrane. In fact, after mitochondrial subfractionation, the main exopolyphosphatase activity was recovered in the membrane

Fig. 6. Polyphosphate as a source for ATP synthesis. Oxygen consumption was monitored using a reaction buffer in the absence of a phosphate source in the eggs on the ninth day of development. The addition of 1 mM ADP, 5 mM pyruvate, 0.5 µM polyphosphate3 and 15, 20 µg/mL heparin, 5 mM phosphate and 0.5 µM oligomycin is represented in the figure. This experiment was repeated at least three times with different preparations, and this figure

phosphate and the energy demand.

fraction, supporting this hypothesis (Table 2).

shows a representative experiment.

Fig. 5. In (A), Mitochondrial exopolyphosphatase activity in *R. microplus* embryos. Mitochondria from eggs on the ninth day of embryogenesis were isolated and exoolyphosphatase activity was determined using polyphosphate3 in the presence of 0.1–2 mM NADH, ADP and Pi. The results represent the mean ±SD of three independent experiments, in triplicate. B) Exopolyphosphatase activity was measured in the mitochondria of eggs on the ninth day of development during mitochondrial respiration with pyruvate as the oxidative substrate, polyphosphate3 as the exopolyphosphatase substrate and olygomicin as ATP synthase. The activity is expressed as units per milligram of total protein and the results represent the mean ± SD of three independent experiments, in triplicate. The asterisk (\*) denotes the difference between the populations and the significance was determined by a two-way ANOVA test (Kruskal-Wallis).

**(B)** \* \*

Fig. 5. In (A), Mitochondrial exopolyphosphatase activity in *R. microplus* embryos. Mitochondria from eggs on the ninth day of embryogenesis were isolated and

significance was determined by a two-way ANOVA test (Kruskal-Wallis).

exoolyphosphatase activity was determined using polyphosphate3 in the presence of 0.1–2 mM NADH, ADP and Pi. The results represent the mean ±SD of three independent experiments, in triplicate. B) Exopolyphosphatase activity was measured in the

mitochondria of eggs on the ninth day of development during mitochondrial respiration with pyruvate as the oxidative substrate, polyphosphate3 as the exopolyphosphatase substrate and olygomicin as ATP synthase. The activity is expressed as units per milligram of total protein and the results represent the mean ± SD of three independent experiments, in triplicate. The asterisk (\*) denotes the difference between the populations and the

Thus, during state 3, a balance existed between the release of phosphate by exopolyphosphatase and ATP synthesis, since exopolyphosphatase activity was measured by the amount of phosphate present. The exopolyphosphatase activity increased during mitochondrial respiration when pyruvate and ADP were added. This increase did not occur without the addition of ADP, indicating that exopolyphosphatase is stimulated during state 3 and that the rate of phosphate release is higher than the rate of ATP synthesis. Indeed, the stimulatory effect was antagonized by olygomicin, an ATP synthase inhibitor (Figure 5B). These data suggest that mitochondrial exopolyphosphatase activity is regulated by phosphate and the energy demand.

Furthermore, it was possible to measure ADP-dependent mitochondrial oxygen consumption in the presence of polyphosphate and in the absence of any other phosphate source. This oxygen consumption was observed using polyphosphate3 and polyphosphate15; however, the consumption was higher with polyphosphaste3. On the other hand, heparin, an exopolyphosphatase inhibitor, blocked oxygen consumption, which was recovered when 5 mM phosphate was added and was again interrupted by the addition of oligomycin, an ATP-synthase inhibitor (Figure 6). These results suggest that polyphosphate was used as a phosphate donor for ATP synthesis due to the mitochondrial coupling observed when mitochondrial respiration was interrupted by oligomycin and the existence of membrane exopolyphosphatase in this process, due to the inhibition by heparin, which cannot cross the mitochondrial membrane and has its active site oriented toward the external face of the membrane. In fact, after mitochondrial subfractionation, the main exopolyphosphatase activity was recovered in the membrane fraction, supporting this hypothesis (Table 2).

Fig. 6. Polyphosphate as a source for ATP synthesis. Oxygen consumption was monitored using a reaction buffer in the absence of a phosphate source in the eggs on the ninth day of development. The addition of 1 mM ADP, 5 mM pyruvate, 0.5 µM polyphosphate3 and 15, 20 µg/mL heparin, 5 mM phosphate and 0.5 µM oligomycin is represented in the figure. This experiment was repeated at least three times with different preparations, and this figure shows a representative experiment.

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks 151

Fig. 7. The effect of some reagents on membrane exopolyphosphatase activity.

mM K+, 10–100 µM molybdate, 1–10 mM NaF and 20µg/mL heparin.

Mitochondrial membrane fractions of *R. microplus* embryos in eggs on the ninth day of embryogenesis were isolated and the membrane exopolyphosphatase activity was

determined using polyphosphate3 as the substrate in the presence of 2.5 mM Mg2+ , 50–200

Fig. 8. Involvement of membrane exopolyphosphatase in mitochondrial respiration. Oxygen consumption was monitored using a reaction buffer in the absence of a phosphate source in the eggs on the ninth day of development in the presence of 1 mM ADP, 5 mM pyruvate,

and 0.5 μM polyphosphate3 and 15. The results represent the mean ± SD of three

independent experiments, in triplicate.


Table 2. Exopolyphosphatase activity in mitochondrial preparations. Exopolyphosphatase activity was measured using eggs on the ninth day of development using polyphosphate3 as the substrate. The activity is expressed as units per milligram of total protein and the results represent the mean ± SD of three independent experiments, in triplicate.
