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Localization of hydrogenase in Desulfovibrio gigas cells

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Abstract

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.

Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.

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Author notes

  1. Vincent Nivière

    Present address: Department of Biological Sciences, The University of Calgary, T2N 1N4, Calgary, Alberta, Canada

Authors and Affiliations

  1. Laboratoire de Chimie Bactérienne, CNRS, F-13277, Marseille Cedex 9, France

    Vincent Nivière, Nicole Forget & Claude E. Hatchikian

  2. Centre de Biochimie et de Biologie Moléculaire, CNRS, F-13277, Marseille Cedex 9, France

    Alain Bernadac

  3. Instituto de Catalisis, CSIC, Serrano 119, E-28006, Madrid, Spain

    Victor M. Fernández

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  1. Vincent Nivière
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  2. Alain Bernadac
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  3. Nicole Forget
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  4. Victor M. Fernández
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  5. Claude E. Hatchikian
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Nivière, V., Bernadac, A., Forget, N. et al. Localization of hydrogenase in Desulfovibrio gigas cells. Arch. Microbiol. 155, 579–586 (1991). https://doi.org/10.1007/BF00245353

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  • DOI: https://doi.org/10.1007/BF00245353

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