A mitochondrial membrane exopolyphosphatase is modulated by, and plays a role in, the energy metabolism of hard tick Rhipicephalus (Boophilus) microplus embryos

Abstract

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.

Keywords: arthropod; energy metabolism; inorganic polyphosphate; membrane exopolyphosphatase; respiration.

Figure 1

Involvement of membrane PPX in mitochondrial respiration. Oxygen consumption was monitored using a reaction buffer in the absence of a P_i source in eggs on the 9th day of development. In (A) and (B), the addition of 1 mM ADP, 5 mM pyruvate, 0.5μM polyP_{3 and 15}, 20 μg/mL heparin, 5 mM P_i 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. In (C), the oxygen consumption was quantified using 1 mM ADP, 5 mM P_i, 5 mM pyruvate and 0.5μM polyP_{3 and 15}. Asterisk (*) denotes the difference between population and the significance was determined by two way ANOVA test (Kruskal–Wallis).

Figure 2

PPX activity in mitochondrial preparations. PPX activity was measured in mitochondria (black bar), soluble (red bar) and membrane fractions (blue bar) of the eggs on the 9th day of development using polyP₃ as substrate. The activity was expressed as units per milligram of total protein and the results represent mean ± SD. of three independent experiments, in triplicate.

Figure 3

Regulation of mitochondrial PPX activity during mitochondrial respiration. PPX activity was measured in mitochondria of the eggs on the 9th day of development during mitochondrial respiration, using pyruvate as oxidative substrates, polyP₃ as PPX substrate, KCN as inhibitor of the respiratory chain, FCCP as uncoupler and Heparin as PPX inhibitor. The activity was expressed as units per milligram of total protein and the results represent mean ± SD. of three independent experiments, in triplicate. Asterisk (*) denotes the difference between population and the significance was determined by two way ANOVA test (Kruskal–Wallis).

Figure 4

Redox regulation of membrane mitochondrial PPX. PPX activity was measured in mitochondria of the eggs on the 9th day of development using polyP₃ as substrate. In (A), the activity was measured in the presence of 0.1–1.0 mM of DTT (■) and H₂O₂ (●). In (B), the mitochondria were treated with 1 mM DTT (black bar) and 1 mM H₂O₂ (hachured bar) for 0–20 min. The results represent mean ± SD. of three independent experiments, in triplicate.