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Citrate lyase from Rhodopseudomonas gelatinosa: Purification, electron microscopy and subunit structure

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Abstract

  1. 1.

    Citrate lyase (EC 4.1.3.6) from Rhodopseudomonas gelatinosa has been purified to homogeneity by protamine sulfate fractionation, chromatography on DEAE-Cellulose and gel filtration. The final enzyme preparation had a specific activity of 138 units per mg of protein and was purified 43-fold over the crude extract. Analysis of citrate lyase by sedimentation equilibrium experiments and gel filtration gave molecular weights of 530000 and 560000, respectively.

  2. 2.

    Electron microscopic investigations of negatively stained enzyme molecules and image analysis showed that citrate lyase is composed of six large and six small subunits; they are arranged in two hexagonal rings lying face to face, each containing, in alternating sequence, three large and three small subunits. The enzyme molecule is 160 Å in diameter and about 100 Å thick.

  3. 3.

    Treatment with sodium dodecylsulfate and mercaptoethanol dissociated citrate lyase into three proteins. Protein III (small subunit) had a molecular weight of 30000 and contained the pantothenate; protein II (large subunit) had a molecular weight of 61000; protein I (M r =97000) was probably an aggregate of II and III.

  4. 4.

    Based on the results obtained a model of citrate lyase was constructed.

  5. 5.

    Purified citrate lyase was obtained from R. gelatinosa in a deacetylated and largely oxidized form. The enzyme was activated by reduction with dithiothreitol (3 mM) and subsequent acetylation with acetic anhydride (1.75 mM).

  6. 6.

    The enzyme was subject to reaction inactivation, the extent of which depended on the concentration of Mg2+.

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Authors and Affiliations

  1. Institut für Mikrobiologie der Universität Göttingen, Göttingen, Germany

    Norbert Beuscher, Frank Mayer & Gerhard Gottschalk

  2. Gesellschaft für Strahlen- und Umweltforschung mbH in Göttingen, Göttingen, Germany

    Norbert Beuscher, Frank Mayer & Gerhard Gottschalk

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  1. Norbert Beuscher
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  2. Frank Mayer
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  3. Gerhard Gottschalk
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Beuscher, N., Mayer, F. & Gottschalk, G. Citrate lyase from Rhodopseudomonas gelatinosa: Purification, electron microscopy and subunit structure. Arch. Microbiol. 100, 307–328 (1974). https://doi.org/10.1007/BF00446325

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  • DOI: https://doi.org/10.1007/BF00446325

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