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: 3.27). 04/2010; 13(1):93-107. DOI: 10.1007/s10126-010-9273-3
Source: PubMed


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 Zn2+ 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


Available from: Zhifeng Zhang, Aug 01, 2014
  • Source
    • "coli) (TransGen Biotech Co., Beijing, China) were transformed with the plasmid pET28a-17␤-HSD8. The recombinant protein, which was primary in inclusion bodies, was expressed in E. coli induced by isopropyl ␤-d-thiogalactoside (IPTG) according to the protocols described by Ma et al. [59]. The inclusion body proteins were collected by centrifugation (10,000 rpm, 4 • C, 10 min), diluted in 8 M urea and purified using Ni 2+ -NTA affinity chromatography (Merck KGaA, Darmstadt, Germany) following manufacturer's instructions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: 17β-hydroxysteroid dehydrogenases (17β-HSDs) are important enzymes catalyzing steroids biosynthesis and metabolism in vertebrates. Although studies indicate steroids play a potential role in reproduction of molluscs, little is known about the presence and function of 17β-HSDs in molluscs. In the present study, a full-length cDNA encoding 17β-HSD type 8 (17β-HSD8) was identified in the Zhikong scallop Chlamys farreri, which is 1,104bp in length with an open reading frame of 759bp encoding a protein of 252 amino acids. Phylogenetic analysis revealed that the C. farreri 17β-HSD8 (Cf-17β-HSD8) belongs to the short chain dehydrogenase/reductase family (SDR) and shares high homology with other 17β-HSD8 homologues. Catalytic activity assay in vitro demonstrated that the refolded Cf-17β-HSD8 expressed in E. coli could effectively convert estradiol-17β (E2) to estrone (E1), and weakly catalyze the conversion of testosterone (T) to androstenedione (A) in the presence of NAD(+). The Cf-17β-HSD8 mRNA was ubiquitously expressed in all tissues analyzed, including gonads. The expression levels of Cf-17β-HSD8 mRNA and protein increased with gametogenesis in both ovary and testis, and were significantly higher in testis than in ovary at growing stage and mature stage. Moreover, results of in situ hybridization and immunohistochemistry revealed that the mRNA and protein of Cf-17β-HSD8 were expressed in follicle cells and gametes at all stages except spermatozoa. Our findings suggest that Cf-17β-HSD8 may play an important role in regulating gametogenesis through modulating E2 levels in gonad of C. farreri.
    Full-text · Article · Jan 2014 · The Journal of steroid biochemistry and molecular biology
  • Source
    • "The midgut tissue and mitochondrial total protein were extracted using the tissue protein extraction kit (Cwbio, Beijing, China). SDS-PAGE and western blotting were carried out as described [21]. A polyclonal antibody of U. unicinctus SDO was prepared by injecting purified recombinant SDO into New Zealand white rabbits at a titer of 1∶ 25,600. "
    [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.
    Full-text · Article · Dec 2013 · PLoS ONE
  • Source
    • "Subsequently, samples were incubated in 3% BSA for 30 min, and then incubated with anti-Aj-VASA antibody (diluted 1:800) for 1 hr. Preparation of the rabbit anti-Aj-VASA antibody was performed as described previously (Ma et al., 2011); the antibody titer was determined by enzyme-linked immunosorbent assay (1:8,000) while the specificity of antibody was tested by Western blotting. Primary antibody was reacted with peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) (diluted 1:5,000) for 1 hr. "
    [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.
    Full-text · Article · Jul 2013 · Molecular Reproduction and Development
Show more