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 (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. 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.

Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mitochondria of Rhipicephalus (Boophilus) microplus Embryo

“…Thereafter, the embryo starts segment formation and initiates organogenesis 24, 54. Previously, we provided evidence that mitochondrial membrane PPX plays a role in energy metabolism in R. microplus during embryo development 18. Here, we demonstrate that inorganic polyphosphate and mitochondrial membrane PPX regulation can be correlated to the generation of reactive oxygen species in mitochondria during R. microplus embryo development.…”

“…Although the first evidence for the presence of poly P in mammalian cells was obtained a long time ago 50, relatively few studies have addressed its physiological roles in animal cells 1, 9-12, 14, 18, 42, 51, 52.…”

“…Mitochondria from tick embryos were previously characterized by our research group 18, 42, 51. Mitochondria from tick embryos were isolated and the mitochondrial hexokinase activity was analyzed during R. microplus embryogenesis.…”

“…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 mitochondrial membrane fraction and the pellet was re-suspended in a buffer containing 10 mM Tris HCl, pH 7.418. Protein was determined by the Folin-Lowry method using bovine serum albumin as a standard 43.…”

“…The interest in mitochondrial poly P is focused on two aspects: poly P as a macroenergetic compound with the same energy of hydrolysis of the phosphoanhydride bond as ATP, and the fact that, according to the endosymbiotic theory, mitochondria originated from ancient prokaryotic cells; thus, it would be intriguing to discover whether or not mitochondria have preserved polyphosphate functions such as the regulation of energy metabolism 16, 17. Recently, we have demonstrated that electron flux and redox states may exert some influence and be influenced by the activity of membrane exopolyphosphatase (PPX), the enzyme that splits P i off the end of a poly P chain, suggesting that it plays a role in energy supply during R. microplus embryogenesis 18.…”