Article

Sulfide:quinone Oxidoreductase from Echiuran Worm Urechis unicinctus

Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, 266003, China.
Marine Biotechnology (Impact Factor: 3.15). 04/2010; 13(1):93-107. DOI: 10.1007/s10126-010-9273-3
Source: PubMed

ABSTRACT Sulfide is a natural, widely distributed, poisonous substance, and sulfide:quinone oxidoreductase (SQR) has been identified to be responsible for the initial oxidation of sulfide in mitochondria. In this study, full-length SQR cDNA was cloned from the echiuran worm Urechis unicinctus, a benthic organism living in marine sediments. The protein consisted of 451 amino acids with a theoretical pI of 8.98 and molecular weight of 50.5 kDa. Subsequently, the SQR mRNA expression in different tissues was assessed by real-time reverse transcription and polymerase chain reaction and showed that the highest expression was in midgut, followed by anal sacs and coelomic fluid cells, and then body wall and hindgut. Furthermore, activated SQR was obtained by dilution refolding of recombinant SQR expression in E. coli, and the refolded product showed optimal activity at 37 °C and pH 8.5 and K (m) for ubiquinone and sulfide at 15.6 µM and 40.3 µM, respectively. EDTA and GSH had an activating effect on refolded SQR, while Zn(2+) caused decreased activity. Western blot showed that SQR in vivo was located in mitochondria and was ∼ 10 kDa heavier than the recombinant protein. In addition, SQR, detected by immunohistochemistry, was mainly located in the epithelium of all tissues examined. Ultrastructural observations of these tissues' epithelium by transmission electron microscopy provided indirect cytological evidence for its mitochondrial location. Interesting aspects of the U. unicinctus SQR amino acid sequence, its catalytic mechanism, and the different roles of these tissues in sulfide metabolic adaptation are also discussed.

Download full-text

Full-text

Available from: Zhifeng Zhang, Aug 01, 2014
0 Followers
 · 
94 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sulfide is a common toxin to animals and is abundant in coastal and aquatic sediments. Sulfur dioxygenase (SDO) is thought to be the key enzyme involved in sulfide oxidation in some organisms. The echiuran worm, Urechis unicinctus, inhabits coastal sediment and tolerates high concentrations of sulfide. The SDO is presumably important for sulfide tolerance in U. unicinctus. The full-length cDNA of SDO from the echiuran worm U. unicinctus, proven to be located in the mitochondria, was cloned and the analysis of its sequence suggests that it belongs to the metallo-β-lactamase superfamily. The enzyme was produced using an E. coli expression system and the measured activity is approximately 0.80 U mg protein(-1). Furthermore, the expression of four sub-segments of the U. unicinctus SDO was accomplished leading to preliminary identification of functional domains of the enzyme. The identification of the conserved metal I (H113, H115, H169 and D188), metal II (D117, H118, H169 and H229) as well as the potential glutathione (GSH) (R197, Y231, M279 and I283) binding sites was determined by enzyme activity and GSH affinity measurements. The key residues responsible for SDO activity were identified by analysis of simultaneous mutations of residues D117 and H118 located close to the metal II binding site. The recombinant SDO from U. unicinctus was produced, purified and characterized. The metal binding sites in the SDO were identified and Y231 recognized as the mostly important amino acid residue for GSH binding. Our results show that SDO is located in the mitochondria where it plays an important role in sulfide detoxification of U. unicinctus.
    PLoS ONE 12/2013; 8(12):e81885. DOI:10.1371/journal.pone.0081885 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sulphides are naturally occurring and widely distributed, poisonous substances and sulphide:quinone oxidoreductase (SQR) has been identified to be responsible for the initial oxidation of sulphide in mitochondria. In a previous study, we found that in the sulphide-adapted species Urechis unicinctus, SQR mRNA concentrations in the mid-gut and in the anal sacs were higher than in the body wall and in the hindgut. To investigate the function of the mid-gut and anal sacs and mitochondrial sulphide metabolism in U. unicinctus, we determined the SQR protein expression in different tissues and the SQR protein expression and enzyme activity after sulphide exposure (25, 50 and 150 µM) in the anal sacs and in the mid-gut. The results showed the highest SQR expression was in the anal sacs, followed by the body wall and the hindgut, and finally the mid-gut. During exposure to 50 µM sulphide, the SQR expression in the anal sacs was significantly increased up to 2 h, reaching a maximum at 24 h and then decreasing up to 48 h. In the anal sacs, SQR enzyme activity was increased significantly up to 6 h and continued to 48 h during exposure to 50 µM sulphide, whereas in mid-gut, the SQR expression and enzyme activity did not increase significantly. We conclude that the anal sacs act as an important organ while the mid-gut only acts as an ‘assistant’ organ for mitochondrial sulphide metabolism in U. unicinctus.
    Marine Biology Research 12/2012; 8(10). DOI:10.1080/17451000.2012.707320 · 1.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Vasa has been extensively used as a germline marker to trace the origin and migration pathway of primordial germ cells (PGCs) in many organisms, but little work has been reported on vasa genes and the origin of PGCs in holothurians. Using in situ hybridization and immunohistochemistry, vasa mRNA and protein of the sea cucumber Apostichopus japonicus (Aj-vasa) was detected in the cytoplasm of the unfertilized egg and was equally distributed in the cytoplasm of early embryos, from the 2-cell embryo to the blastula, indicating that Aj-vasa mRNA is maternally supplied. Later, expression of both Aj-vasa mRNA and protein centralizes gradually in newly organized structures from blastula to five-tentacle larva, and then is restricted to PGC-like cells of the original gonad in juveniles with 0.1-cm body length. The structure of the gonad develops further from a simple tubular gonad in 0.5-cm-length juveniles to a branched gonad in 3-cm-length juveniles. Our findings showed that the maternal supply of the vasa gene products in A. japonicus is different from that in sea urchin Strongylocentrotus purpuratus, of echinoderm, and suggested that the specialization of PGCs is an epigenesis mechanism in A. japonicus. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
    Molecular Reproduction and Development 07/2013; DOI:10.1002/mrd.22207 · 2.68 Impact Factor