Mitochondria from tick embryos in the segmentation stage (9th day after oviposition) were isolated and cellular respiration was measured using pyruvate as the substrate. Oxygen consumption was 30 nmol/min·mg protein and the RCR (respiratory control ratio) was 6.5. The process was KCN- and oligomycin-sensitive (Table 1).

Respiratory parameters of mitochondria in the presence of pyruvate (5 mM). The rates of respiration in State 3 (phosphorylating respiratory rate) and in State 4 (non-phosphorylating respiratory rate) are expressed as nmol O₂/min·mg protein. The results represent mean ± SD. of three independent experiments.

We have previously demonstrated that polyP can be used as a P_i donor for adenosine-5′-triphosphate (ATP) synthesis in ticks [15]. To investigate the location of mitochondrial PPX, we assayed oxygen consumption using polyP₃ and polyP₁₅ as substrates and heparin as a PPX inhibitor. adenosine diphosphate-dependent mitochondrial oxygen consumption could be measured in the presence of polyP₃ and polyP₁₅ and in the absence of any other source of P_i, supporting the hypothesis previously postulated that polyP can be used as a P_i donor for ATP synthesis [15]. However, this consumption was inhibited by heparin. Subsequently, oxygen consumption was recovered when 5 mM P_i was added, which was interrupted by addition of oligomycin, an ATP-synthase inhibitor (Figure 1A and 1B). Oxygen consumption was distinct using both polyP: no statistical difference was found using polyP₃ compared with P_i; otherwise, oxygen consumption was lower when polyP₁₅ was used (Figure 1C).

This new data suggest the existence of membrane PPX in this process due to the inhibition by heparin, which cannot cross the mitochondrial membrane, which has its active site oriented to the external face of the membrane. In fact, after subfractionation, the main PPX activity was recovered in the membrane fraction, supporting this hypothesis (Figure 2).

Exopolyphosphatases have been found in prokaryotes and eukaryotes, and although in bacteria these enzymes hydrolyze mostly high-molecular-weight polyP [17], at least some of the enzymes from S. cerevisiae and Leishmania major are more active in hydrolyzing short-chain polyP such as polyP₃ [17,30].

To obtain insight into membrane PPX kinetics, the apparent K_m was measured using polyP₃ and polyP₁₅ as substrates and results were expressed as the average of three independent experiments. Parameters obtained were very similar to those observed in crude mitochondria recently for our group [16]; the membrane PPX affinity for polyP₃ was 10 times stronger than for polyP₁₅ (Table 2). These results are in contrast to those found in a mitochondria membrane-bound PPX of S. cerevisiae, in which case the affinity was stronger for long-chain polyP [31]. However, the new data demonstrated that membrane PPX kinetics are in agreement with oxygen consumption that was much higher using polyP₃ than polyP₁₅. Heparin, an effective inhibitor of PPX [1], blocked the activity (Table 2). These results reinforce the coupling existing between this enzyme activity and mitochondrial ADP phosphorylation.

To further investigate regulation of membrane PPX during mitochondrial respiration, the activity was measured using pyruvate as the substrate and polyP as the only source of P_i. During this assay, addition of a small amounts of ADP (0.2 mM) induces a state 3 followed by a state 4, when all the ADP was converted to ATP. Thus, during state 3, a balance exists between P_i release by PPX and ATP synthesis because PPX is measured by the amount of P_i. Membrane PPX activity increased during mitochondrial respiration when pyruvate and ADP were added. This increase did not occur without ADP addition, indicating that PPX is stimulated during state 3 and the velocity of P_i release is higher than ATP synthesis. Indeed, the stimulatory effect was antagonized by KCN (decreased electron flux) addition and increased by FCCP (increased electron flux) (Figure 3), suggesting that membrane PPX could be modulated by electron flux. These data are in agreement with our previous work in which we demonstrated that mitochondrial PPX activity is regulated by energy demand [15]. Additionally, these findings are consistent with those of [32], who demonstrated that production and consumption of mitochondrial polyP depend on the activity of the oxidative phosphorylation machinery in mammalian cells. Furthermore, heparin inhibited PPX activity completely, reinforcing the role of membrane PPX during mitochondrial respiration and the respiration activation by membrane PPX activity indicates that PPX could be close to the site of ATP production.

Additionally, mitochondrial polyP can form polyP/Ca²⁺/PHB complexes [3] with ion-conducting properties similar to those of the native mitochondrial permeability transition pore [25]. Polyphosphatases localized in the membrane may not only degrade, but also synthesize polyP inside these complexes [31]. Recently, we demonstrated that synthesis of polyP occurs during embryogenesis of R. microplus in mitochondria, but not in nuclei [15,16]. As polyphosphate kinases have been found only in prokaryotes, the observation that polyP synthesis in ticks only occurs in the mitochondrial fraction supports the possibility that such synthesis probably occurs by the action of these complexes, as already suggested for other organisms [3,31,33,34].

Despite regulation of membrane PPX by increased or decreased electron flux, the sensitivity of this enzyme according to redox state using polyP₃ as the substrate was evaluated. The influence of dithiothreitol (DTT) and hydrogen peroxide (H₂O₂) was investigated at different times and in a concentration range of 0.1 to 1.0 mM and PPX activity was stimulated and inhibited by 50%, respectively, suggesting that PPX is tightly regulated by redox state (Figure 4A and 4B).

Ticks were obtained from a colony maintained at the Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil. R. microplus (Acarina, Ixodidae) ticks from the Porto Alegre strain, free of parasites, were reared on calves obtained from a tick-free area. Engorged adult females were maintained in Petri dishes at 28 °C and 80% relative humidity upon completion of oviposition, which starts approximately three days after adult ticks drop off calves. Animals were treated in compliance with the UFRGS review committee for animal care.

ADP, pyruvate, sodium phosphate glass type 15 (polyP₁₅), sodium tripolyphosphate (polyP₃), heparin, FCCP, oligomycin, KCN, DTT, H₂O₂ were purchased from Sigma Adrich. All other reagents were analytical grade.

The cell fractionation procedure used required large amounts of fresh eggs (at least 2 g) to obtain functionally active mitochondrial fractions. For characterization of mitochondrial fractions, eggs in the segmentation stage (9th day after oviposition) were used and mitochondria were isolated as previously described [15]. Isolation of the mitochondria membrane fraction was performed by sonication of freshly prepared mitochondria three times for 20 s at the maximal output using an MSE ultrasonic disintegrator. The suspension was centrifuged for 10 min at 12,000 × g to remove unbroken mitochondria. The supernatant was centrifuged at 100,000 × g for 60 min to yield the mitochondria membrane fraction. The supernatant was a soluble preparation of mitochondria, which included both the intermembrane space and matrix, and the pellet was a mix of inner and outer membranes.

The reaction mixture consisted of 50 mM Tris-HCl buffer (pH 7.5) and 5 mM MgCl₂, using 5 mM polyP₃ or polyP₁₅, as the substrate. Reactions were performed at 30 °C for various time periods. The P_i formed during the reaction was spectrophotometrically determined as previously described [35] by adding a solution of 0.5% ammonium molybdate, 0.35 M sulfuric acid, 0.5% sodium dodecyl sulfate, and 10% ascorbic acid. Measurements of absorbance at 750 nm were performed after 15 min. The enzyme amount liberating 1 μmoL of P_i per 1 min was defined as one unit of enzyme activity (U). Protein concentration was measured as described previously [36] using bovine serum albumin as a standard.

PPX activity during mitochondrial respiration was measured using a reaction mixture consisting of 50 mM Tris-HCl buffer (pH 7.2), 120 mM KCl, 1 mM EGTA, 5 mM MgCl₂, and 0.2 mM adenosine diphosphate (ADP) in the absence of any P_i source. PolyP₃ (0.5 μM) was used as a substrate for PPX activity and 5 mM pyruvate was used as an oxidative substrate. Potassium cyanide (KCN, 1 mM) and 20 μg/mL heparin were used to inhibit cytochrome oxidase and PPX activities, respectively, and 200 nM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was used as an uncoupler. The reaction was performed at 28 °C for 15 min [15].

Kinetic parameters were estimated by nonlinear regression analysis applied to the Michaelis-Menten equation using the program package supplied by GraphPad Prism 5.0.

The rate of O₂ uptake by mitochondria was estimated with a Clark oxygen electrode (YSI, mod. 5775, Yellow Springs, OH, USA). The calibration process was conducted using the initial O₂ concentration of the medium as 100% O₂-saturated buffer measured at 28 °C. Measurements were performed in 1.5 mL of reaction buffer containing 120 mM KCl, 1 mM EGTA, 0.2% bovine albumin, and 3 mM HEPES (pH 7.2) in the absence of any P_i source, containing 0.5 mg/mL of mitochondrial protein. After a 1-min equilibration period, mitochondrial respiration was started by addition of pyruvate to a final concentration of 5 mM. Each experiment was repeated at least three times with different mitochondrial preparations. Figure 1 shows a representative experiment and other additions are indicated in the figure legend [15]. Respiratory control ratio (RCR) values were obtained with isolated mitochondria by using pyruvate as the complex I substrate.