Int. J. Biol. Sci. 2013, Vol. 9 842 Ivyspring International Publisher International Journal of Biological Sciences 2013; 9(8):842-852. doi: 10.7150/ijbs.6628 Research Paper Inorganic Polyphosphates Regulate Hexokinase Activity and Reactive Oxygen Species Generation in Mito- chondria of Rhipicephalus (Boophilus) microplus Embryo Amanda Fraga1, Jorge Moraes1,2, José Roberto da Silva1,2, Evenilton P. Costa3, Jackson Menezes1,2, Itabajara da Silva Vaz Jr2,4, Carlos Logullo2,3, Rodrigo Nunes da Fonseca 1,2 and Eldo Campos1,2 1. Laboratório Integrado de Bioquímica—Hatisaburo Masuda, UFRJ, Polo Barreto, Av. São José do Barreto nº 764, São Jose do Barreto, CEP 27971-550, Macaé, RJ, Brazil; 2. Instituto Nacional de Ciência e Tecnologia—Entomologia Molecular, Rio de Janeiro, RJ, CEP 21941–590, Brazil; 3. Laboratório de Química e Função de Proteínas e Peptídeos and Unidade de Experimentação Animal–CBB–UENF, Avenida Alberto Lamego, 2000, Horto, Campos dos Goytacazes, RJ, CEP 28015–620, Brazil; 4. Centro de Biotecnologia e Faculdade de Veterinária, UFRGS, Avenida Bento Gonçalves, 9090, Porto Alegre, RS, C.P. 15005, CEP 91501–970, Brazil. Corresponding author: Tel.: +55-22-33993967. E-Mail address: [email protected] (E. Campos). © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2013.05.06; Accepted: 2013.08.08; Published: 2013.08.27 Abstract The physiological roles of polyphosphates (poly P) recently found in arthropod mitochondria remain obscure. Here, the possible involvement of poly P with reactive oxygen species generation in mitochondria of Rhipicephalus microplus embryos was investigated. Mitochondrial hexokinase and scavenger antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione re- ductase were assayed during embryogenesis of R. microplus. The influence of poly P3 and poly P15 were analyzed during the period of higher enzymatic activity during embryogenesis. Both poly Ps inhibited hexokinase activity by up to 90% and, interestingly, the mitochondrial membrane exo- polyphosphatase activity was stimulated by the hexokinase reaction product, glucose-6-phosphate. Poly P increased hydrogen peroxide generation in mitochondria in a situation where mitochondrial hexokinase is also active. The superoxide dismutase, catalase and glutathione reductase activities were higher during embryo cellularization, at the end of embryogenesis and during embryo segmentation, respectively. All of the enzymes were stimulated by poly P3. However, superoxide dismutase was not affected by poly P15, catalase activity was stimulated only at high concentrations and glutathione reductase was the only enzyme that was stimulated in the same way by both poly Ps. Altogether, our results indicate that inorganic polyphosphate and mitochondrial membrane exopolyphosphatase regulation can be correlated with the generation of reactive oxygen species in the mitochondria of R. microplus embryos. Key words: inorganic polyphosphate; reactive oxygen species; arthropod; mitochondria; scavenger antioxidant enzymes. Introduction Inorganic polyphosphates (poly P) are long have been demonstrated to play diverse roles de- chains of a few to several hundred phosphate residues pending on the cell type and circumstances [1, 2]. The linked by phosphoanhydride bonds. Polyphosphates biological roles played by polyphosphates have been have been found in all cell types examined to date and most extensively studied in prokaryotes and unicel- http://www.ijbs.com Int. J. Biol. Sci. 2013, Vol. 9 843 lular eukaryotes, where they have been shown to enzyme is present in the matrix (Mn-SOD) and in the regulate many biochemical processes including the intermembrane space (Zn-SOD) [29-32]. The abun- metabolism and transport of inorganic phosphate, dance of this enzyme, as well as its presence in both cation sequestration and storage [1], membrane mitochondrial compartments, attests to the im- channel formation [3, 4], cell envelope formation and portance of removing mitochondrially-generated •__ bacterial pathogenesis[5, 6], regulation of gene and O2 [33]. Catalase (CAT) acts by decomposing H2O2 enzyme activities [7] and activation of Lon proteases into H2O and O2, avoiding the production of hydroxyl [8]. radicals (HO•) in the presence of transition metals. Conversely, poly P functions have not been ex- The presence of CAT in mitochondria is of great im- tensively investigated in higher eukaryotes, although portance, as decomposition of H2O2 protects these some functions have been described such as the acti- organelles against intra- and extra-mitochondrially vation of TOR kinase [9], involvement in blood coag- generated H2O2 [34, 35]. Glutathione reductase (GR) is ulation [10], and apoptosis [11-13]. Regarding mito- the enzyme that regenerates reduced glutathione in chondrial metabolism, mammalian mitochondrial the mitochondrial matrix. Reduced glutathione can poly P production is directly linked to their energetic scavenge ROS non-enzymatically or by serving as an state [14], as the level of poly P regulates the level of electron-donating substrate to several enzymes in- cellular ATP [14] and Ca2+ accumulation [15]. The volved in ROS-detoxification [36]. interest in mitochondrial poly P is focused on two Ticks are vectors of parasites that cause hemo- aspects: poly P as a macroenergetic compound with parasitic diseases and are endemic in many cattle the same energy of hydrolysis of the phosphoanhy- production areas [37]. The present study focuses on dride bond as ATP, and the fact that, according to the Rhipicephalus microplus, which causes heavy economic endosymbiotic theory, mitochondria originated from losses to bovine herds, particularly in tropical regions; ancient prokaryotic cells; thus, it would be intriguing thus, major efforts have been directed toward devel- to discover whether or not mitochondria have pre- oping immunoprophylactic tick-control tools [38, 39]. served polyphosphate functions such as the regula- R. microplus has only one host throughout its three life tion of energy metabolism [16, 17]. Recently, we have stages, which is usually a bovine, and has a long demonstrated that electron flux and redox states may feeding period (approximately 21 days). Female ticks, exert some influence and be influenced by the activity after engorgement, drop off the host and initiate ovi- of membrane exopolyphosphatase (PPX), the enzyme position approximately three days later. Being an that splits Pi off the end of a poly P chain, suggesting oviparous animal, embryogenesis occurs in the ab- that it plays a role in energy supply during R. mi- sence of exogenous nutrients; maternal nutrients are croplus embryogenesis [18]. packaged into oocytes and stored mostly as yolk The metabolism of free hexoses begins by granules. Hatching occurs approximately 21 days phosphorylation in a reaction catalyzed by the hexo- after egg laying and the emerging larvae can survive kinase (ATP: hexose-6-phosphotransferase, E.C. for several weeks before finding a host, using the re- 2.7.1.1; HK). This enzyme has been characterized in maining yolk as their only energy source [40, 41]. several organisms [19-24]. Four distinct hexokinase The aim of this study was to investigate the pos- isozymes are reported for mammalian tissues and are sible involvement of poly P in reactive oxygen species named types I–IV. These isozymes have an internal generation in mitochondria of R. microplus embryos. repeat sequence in their N- and C- terminal halves Such findings could reveal an important role for poly that is found in mammals, insects and nematodes [20, P metabolism in arthropods. 25, 26]. In arthropods the binding mechanism of HK to the outer mitochondrial membrane is not fully es- Materials and Methods tablished. Ticks and reagents The mitochondrial electron transport system (ETS) represents one of the major sources of cellular Ticks were obtained from a colony maintained at reactive oxygen species (ROS) such as superoxide the Faculdade de Veterinária, Universidade Federal __ do Rio Grande do Sul (UFRGS), Brazil as previously (O2• ) and hydrogen peroxide (H2O2), providing continuous generation of these toxic products [27]. described [39]. R. microplus (Acarina, Ixodidae) ticks The scavenger activity of antioxidant enzymes con- from the Porto Alegre strain, free of parasites, were stitutes an essential mechanism directed against the reared on calves obtained from a tick-free area. En- primary ROS generated by mitochondrial ETS. Su- gorged adult females were maintained in Petri dishes •__ at 28°C and 80% relative humidity upon completion peroxide dismutase (SOD) rapidly converts O2 to of oviposition, which starts approximately three days H2O2, protecting the mitochondrial iron-sulfur clus- •__ after adult ticks drop off calves. Animals were treated ter-containing enzymes from O2 attack [28]. This http://www.ijbs.com Int. J. Biol. Sci. 2013, Vol. 9 844 in compliance with the UFRGS review committee for 37°C. The Pi formed during the reaction was spec- animal care. The reagents were purchased from Sigma trophotometrically determined as described by[44], (USA), Amersham Biosciences (USA), Invitrogen adding a solution of 0.5% ammonium molybdate, 0.35 (USA) and Merck (Germany). M sulfuric acid, 0.5% sodium dodecyl sulfate, and 10%