| Sulfide (the sum of H2S, HS- and S2-) is a well known and widely distributed toxin that inhibits many enzymes involved in aerobic metabolism including the mitochondrial terminal oxidase, cytochrome c oxidase (CCO) increasing the free radical production by inducing reactive oxygen species (ROS) and even leading to death. Nevertheless, a variety of marine invertebrates living in habitats such as hydrothermal vents hydrocarbon seeps, coastal mudflats and marshes encountering periodically or continuously exposure of sulfide, can survive by the sulfide metabolism. Urechis unicinctus is a benthic organism, mostly living in sulfidic habitats including intertidal and subtidal zones. Previous study has proved the worm has strong sulfide endurance and detoxification ability. In this study, the main emphasis of our research has focused to the alternative oxidase (AOX) , a ubiquinol oxidase, introducing a branch point in to the respiratory electron transport chain (ETC), was thought to play a crucial by regulate the redox in animal sulfide oxidation. We cloned the full-length cDNA of U.unicinctus AOX, and the AOX mRNA expression pattern coupled with the activity of cytochrome c oxidase (CCO) in body wall and hindgut were analyzed. Our aim is to reveal the AOX expression characteristics under sulfide exposure, and extend the understanding of the regulation of mitochondria sulfide detoxification in animals. This work is also expected to provide useful information for further investigation on the functions of AOX.The full-length of U.unicinctus AOX cDNA was cloned, The complete sequence of AOX cDNA consisted of a 5'terminal untranslated region (UTR) of 288 bp, a 3'UTR of 390 bp, and an open reading frame (ORF) of 1047 bp which contains a termination codon (TAA) and encodes a putative protein of 348 amino acids with a molecular weight of 39.49 kDa and a theoretical isoelectric point (pI) of 8.49. The deduce protein contained conserved LET, NERMHL, LLEEA, RADE_ _H regions and a Q-binding site, which are the main characteristic features of AOX. TargetP analysis showed that U. unicinctus AOX was located in the mitochondria, with a score of 0.673; topology prediction for the 348 amino acids by TMHMM showed the protein have two transmembrane helices (TMHelix I 170-192 aa and TMHelix II 233-255 aa)The mRNA expression pattern combined activity of cytochrome c oxidase (CCO) in bodywall and hindgut under sulfide exposure (50μM and 150μM) were measured. The results revealed AOX mRNA expression was increased in a time- and concentration-dependent manner in both tissues, significantly increased at 48 h and continuously increased with time.Meanwhile, the activities of CCO were decreased when reached the max at 6 h and were complete inhibited 48 h after exposure under high concentration sulfide (150μM).The present data show a negative relationship of change trends between the expression of AOX mRNA and activity of CCO. And our study indicates that AOX play an important role in mitochondria sulfide detoxification. |
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