**2.1.1 A mitochondrial membrane exopolyphosphatase**

Exopolyphosphatases have been found in prokaryotes and eukaryotes and, although in bacteria these enzymes mostly hydrolyze high molecular weight polyphosphates (Kumble & Kornberg, 1996), at least some of the enzymes from *Saccharomyces cerevisiae* and *Leishmania major* are more active in hydrolyzing short chain polyphosphates, such as polyphosphate3 (Kumble & Kornberg, 1996; Rodrigues et al., 2002). Exopolyphosphatase from *Escherichia coli* requires divalent cations and K+ for maximum activity, while exopolyphosphatase from yeast only requires divalent cations (Lichko et al., 2003). Membrane mitochondrial exopolyphosphatase activity from the hard tick *R. microplus* was found to be stimulated by Mg2+ and was insensitive to K+. Only a few compounds that inhibit exopolyphosphatase have been identified (Kornberg et al., 1999): treatment with molybdate (a common phosphohydrolase inhibitor) and fluoride (a pyrophosphatase inhibitor) showed that exopolyphosphatase present in the mitochondrial membrane fractions was insensitive to these compounds. However, heparin, a good inhibitor of other well-characterized exopolyphosphatases (Lichko et al., 2003), was effective in almost 100% (Figure 7). In order to obtain an insight into membrane exopolyphosphatase kinetics, the apparent *Km* was measured using polyphosphate3 and polyphosphate15 as substrates and the results were expressed as the average of three independent experiments. The membrane exopolyphosphatase affinity for polyphosphate3 was 10 times stronger than for polyphosphate15 (Table 3). These results are in contrast with those found in a mitochondrial membrane-bound exopolyphosphatase of *Saccharomyces cerevisiae*, in which case the affinity was stronger for long-chain polyphosphates (Lichko et al., 1998). However, the data demonstrated that membrane exopolyphosphatase kinetics were in agreement with the oxygen consumption rate, which was much higher for polyphosphate3 than polyphosphate15. These results reinforce the theory of coupling between the activity of this enzyme and mitochondrial ADP phosphorylation (Figure 8).


Table 3. Kinetics characterization of exopolyphosphatase activity in membrane preparations of mitochondria from *R. microplus* embryos on the ninth day of embryogenesis.

Mitochondria 0.60 ± 0.19 98

(intermembrane space and matrix) 0.35 ± 0.06 <sup>98</sup>

(mixture of inner and outer membranes) 1.11 ± 0.16 <sup>98</sup>

represent the mean ± SD of three independent experiments, in triplicate.

**2.1.1 A mitochondrial membrane exopolyphosphatase** 

enzyme and mitochondrial ADP phosphorylation (Figure 8).

Substrates Km

(µM)

PolyP3 0.2 2.4 PolyP15 2.2 1.1 Table 3. Kinetics characterization of exopolyphosphatase activity in membrane preparations

of mitochondria from *R. microplus* embryos on the ninth day of embryogenesis.

Vmax (µmol·min−1·mg protein−1)

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

Exopolyphosphatases have been found in prokaryotes and eukaryotes and, although in bacteria these enzymes mostly hydrolyze high molecular weight polyphosphates (Kumble & Kornberg, 1996), at least some of the enzymes from *Saccharomyces cerevisiae* and *Leishmania major* are more active in hydrolyzing short chain polyphosphates, such as polyphosphate3 (Kumble & Kornberg, 1996; Rodrigues et al., 2002). Exopolyphosphatase from *Escherichia coli* requires divalent cations and K+ for maximum activity, while exopolyphosphatase from yeast only requires divalent cations (Lichko et al., 2003). Membrane mitochondrial exopolyphosphatase activity from the hard tick *R. microplus* was found to be stimulated by Mg2+ and was insensitive to K+. Only a few compounds that inhibit exopolyphosphatase have been identified (Kornberg et al., 1999): treatment with molybdate (a common phosphohydrolase inhibitor) and fluoride (a pyrophosphatase inhibitor) showed that exopolyphosphatase present in the mitochondrial membrane fractions was insensitive to these compounds. However, heparin, a good inhibitor of other well-characterized exopolyphosphatases (Lichko et al., 2003), was effective in almost 100% (Figure 7). In order to obtain an insight into membrane exopolyphosphatase kinetics, the apparent *Km* was measured using polyphosphate3 and polyphosphate15 as substrates and the results were expressed as the average of three independent experiments. The membrane exopolyphosphatase affinity for polyphosphate3 was 10 times stronger than for polyphosphate15 (Table 3). These results are in contrast with those found in a mitochondrial membrane-bound exopolyphosphatase of *Saccharomyces cerevisiae*, in which case the affinity was stronger for long-chain polyphosphates (Lichko et al., 1998). However, the data demonstrated that membrane exopolyphosphatase kinetics were in agreement with the oxygen consumption rate, which was much higher for polyphosphate3 than polyphosphate15. These results reinforce the theory of coupling between the activity of this

Soluble fraction

Membrane fraction

Exopolyphosphatase activity (U / mg protein)

Heparin (% inhibition)

Fig. 7. The effect of some reagents on membrane exopolyphosphatase activity. 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 mM K+, 10–100 µM molybdate, 1–10 mM NaF and 20µg/mL heparin.

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.

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks 153

Despite the regulation of membrane exopolyphosphatase by an increased or decreased electron flux, the sensitivity of this enzyme according to the redox state using polyphosphate3 as the substrate was evaluated. The influence of 1.0 mM dithiothreitol (DTT) and 1.0 mM hydrogen peroxide (H2O2) was investigated at different times and the exopolyphosphatase activity was stimulated and inhibited by 50% of both, suggesting that

exopolyphosphatase is tightly regulated by the redox state (Figure 10).

Fig. 10. The redox regulation of mitochondrial membrane exopolyphosphatase.

Fig. 11. Polyphosphate quantification in the nuclear and mitochondrial fractions.

fraction (●) during embryogenesis. The results represent the mean ± SD of three

Polyphosphate levels during embryogenesis in the nuclear fraction (■) and mitochondrial

independent experiments, in triplicate.

independent experiments, in triplicate.

Exopolyphosphatase activity was measured in the mitochondria of the eggs on the ninth day of development using polyphosphate3 as the substrate. The mitochondria were treated with 1 mM DTT and 1 mM H2O2 for 0–20 min. The results represent the mean ± SD of three

To further investigate the regulation of membrane exopolyphosphatase during mitochondrial respiration, the activity was measured using pyruvate as the substrate and polyphosphte as the only source of phosphate. Membrane exopolyphosphatase activity increased during mitochondrial respiration when pyruvate and ADP were added and the stimulatory effect was antagonized by potassium cyanide addition (decreased electron flux) and increased by protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (increased electron flux), suggesting that membrane exopolyphosphatase could be modulated by the electron flux (Figure 9). These findings are consistent with those of Pavlov et al., 2010, who demonstrated that the production and consumption of mitochondrial polyphosphate depends on the activity of the oxidative phosphorylation machinery in mammalian cells. Furthermore, heparin completely inhibited exopolyphosphatase activity, reinforcing the role of membrane exopolyphosphatase during mitochondrial respiration, and the respiration activation by membrane exopolyphosphatase activity indicated that exopolyphosphatase could be close to the site of ATP production.

Fig. 9. Regulation of mitochondrial exopolyphosphatase activity during mitochondrial respiration. The activity of exopolyphosphatase was measured in the mitochondria of the eggs on the ninth day of development during mitochondrial respiration, using pyruvate as the oxidative substrate, polyphosphate3 as the exopolyphosphatase substrate, KCN as the respiratory chain inhibitor, FCCP as the un-coupler and heparin as the exopolyphosphatase inhibitor. The activity was 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).

To further investigate the regulation of membrane exopolyphosphatase during mitochondrial respiration, the activity was measured using pyruvate as the substrate and polyphosphte as the only source of phosphate. Membrane exopolyphosphatase activity increased during mitochondrial respiration when pyruvate and ADP were added and the stimulatory effect was antagonized by potassium cyanide addition (decreased electron flux) and increased by protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (increased electron flux), suggesting that membrane exopolyphosphatase could be modulated by the electron flux (Figure 9). These findings are consistent with those of Pavlov et al., 2010, who demonstrated that the production and consumption of mitochondrial polyphosphate depends on the activity of the oxidative phosphorylation machinery in mammalian cells. Furthermore, heparin completely inhibited exopolyphosphatase activity, reinforcing the role of membrane exopolyphosphatase during mitochondrial respiration, and the respiration activation by membrane exopolyphosphatase activity indicated that

\*

Fig. 9. Regulation of mitochondrial exopolyphosphatase activity during mitochondrial respiration. The activity of exopolyphosphatase was measured in the mitochondria of the eggs on the ninth day of development during mitochondrial respiration, using pyruvate as the oxidative substrate, polyphosphate3 as the exopolyphosphatase substrate, KCN as the respiratory chain inhibitor, FCCP as the un-coupler and heparin as the exopolyphosphatase inhibitor. The activity was 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).

exopolyphosphatase could be close to the site of ATP production.

Despite the regulation of membrane exopolyphosphatase by an increased or decreased electron flux, the sensitivity of this enzyme according to the redox state using polyphosphate3 as the substrate was evaluated. The influence of 1.0 mM dithiothreitol (DTT) and 1.0 mM hydrogen peroxide (H2O2) was investigated at different times and the exopolyphosphatase activity was stimulated and inhibited by 50% of both, suggesting that exopolyphosphatase is tightly regulated by the redox state (Figure 10).

Fig. 10. The redox regulation of mitochondrial membrane exopolyphosphatase. Exopolyphosphatase activity was measured in the mitochondria of the eggs on the ninth day of development using polyphosphate3 as the substrate. The mitochondria were treated with 1 mM DTT and 1 mM H2O2 for 0–20 min. The results represent the mean ± SD of three independent experiments, in triplicate.

Fig. 11. Polyphosphate quantification in the nuclear and mitochondrial fractions. Polyphosphate levels during embryogenesis in the nuclear fraction (■) and mitochondrial fraction (●) during embryogenesis. The results represent the mean ± SD of three independent experiments, in triplicate.

Role of Inorganic Polyphosphate in the Energy Metabolism of Ticks 155

Clements, A., Bursa, D., Gatsos, X., Perry, A, J., Civciristov, S., Celik, N.,Likic, V, A., Poggio,

Fagotto, F. (1990). Yolk degradation in tick eggs: I. Occurrence of a cathepsin L-like acid

Guerrero, F, D., Nene, V, M., George, J, E., Barker, S, C. & Willadsen, P. (2006). Sequencing a

Kim, K, S., Rao, N, N., Fraley, C, D. & Kornberg, A. (2002). Inorganic polyphosphate is

Kornberg, A. (1995). Inorganic polyphosphate: toward making a forgotten polymer

Kornberg, A., Rao, N, N. & ult-Riche, D. (1999). Inorganic polyphosphate: a molecule of

Kulaev, I, S. & Vagabov, V, M. (1983). Polyphosphate metabolism in micro-organisms.

Kulaev, I. & Kulakovskaya, T. (2000). Polyphosphate and phosphate pump. *Annual Review of* 

Kulaev, I, S.; Vagabov, V, M. & Kulakovskaya, T, V. (2004). *The Biochemistry of Inorganic* 

Kulakovskaya, T, V., Lichko, L, P., Vagabov, V, M., & Kulaev, I, S. (2010). Inorganic

Kumble, K. D. & Kornberg, A. (1996). Endopolyphosphatases for long chain inorganic

Kuroda, A., Nomura, K., Ohtomo, R., Kato, J., Ikeda, T., Takiguchi, N., Ohtake, H. &

Lichko, L. P., Andreeva, N. A., Kulakovskaya, T. V. & Kulaev, I. S. (2003).

Polyphosphates in Mitochondria. *Biochemistry (Moscow),* Vol.75, (July 2010), pp.

polyphosphate in yeast and mammals. *Journal of Biological Chemistry*, Vol.271,

Kornberg, A. (2001). Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E. coli. *Science,* Vol. 293, (July 2001), pp.

Exopolyphosphatases of the yeast Saccharomyces cerevisiae. *FEMS Yeast Research*,

*Advances in Microbiol Physiology,* Vol.24, pp. 83-171, ISSN 0065-2911

*Microbiology*, Vol. 54, (October 2000), pp. 709-734, ISSN 0066-4227

*Polyphosphate,* Wiley, ISBN 0 470 85810 9, Chichester, England

(October, 1996), pp. 27146–27151, ISSN 0021-9258

Vol.3, (January, 2003), pp. 233–238, ISSN 1567-1356

*Chemistry,* Vol.25, (March, 1956), pp. 350-356, ISSN 0003-2700

(February, 1990), pp. 217-235, ISSN 0739-4462

*Biomembranes*, Vol*.* 1612, pp. 90-97, ISSN 0005-2736

7680, ISSN 0027-8424

825-831, ISSN 0006-2979

705-708, ISSN 0036-8075

0021-9193

0066-4154

S., Jacobs-Wagner, C., Strugnell, R, A. & Trevor Lithgow, T. (2009). The reducible complexity of a mitochondrial molecular machine. *Proceedings of the National Academy of Sciences*, Vol.106, (September 2009), pp. 15791-15795, ISSN 0027-8424 Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956).

Calorimetricmethod for determination of sugar and related substances. *Analytical* 

proteinase in yolk spheres. *Archives of Insect Biochemistry and Physiology,* Vol.14,

new target genome: the *Boophilus microplus* (Acari: Ixodidae) genome project. *Journal of Medical Entomology,* Vol.43, (January, 2006), pp. 9-16, ISSN 0022-2585 Jones, H, E., Holland, I, B., Jacq, I, B., Wall, T. & Campbell, A.K. (2003). Escherichia coli

lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB– polyphosphate Ca2+ channels in the membrane. *Biochimica et Biophysica Acta* 

essential for long-term survival and virulence factors in Shigella and Salmonella spp. *Proceedings of the National Academy of Sciences*, Vol.99, (May 2002), pp. 7675-

unforgettable. *Journal of Bacteriology,* Vol.177, (February, 1995), pp. 491-496, ISSN

many functions. *Annual Review of Biochemistry*, Vol. 68, (July 1999), pp. 89-125, ISSN

Additionally, mitochondrial polyphosphate can form polyphosphate/Ca2+/PHB complexes (Reusch, 1989) with ion-conducting properties similar to those of the native mitochondrial permeability transition pore (Pavlov et al., 2005). Polyphosphatases localized in the membrane can not only degrade, but they can also synthesize polyphosphate inside these complexes (Lichko et al., 1998). During the embryogenesis of *R. microplus*, the synthesis of polyphosphate occurs in mitochondria but not in the nuclei (Figure 11). As polyphosphate kinases have only been found in prokaryotes, the observation that polyphosphate synthesis in ticks only occurs in the mitochondrial fraction supports the possibility that such synthesis probably occurs via the action of these complexes, as already suggested for other organisms (Reusch and Sadoff, 1988; Lichko et al., 1998; Reusch et al., 1998; Abramov et al., 2007).
