Archives of Microbiology

, Volume 123, Issue 2, pp 203–208 | Cite as

NADH-dependent reduction of d-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase, d-proline reductase, and a third protein factor

  • Arnold C. Schwartz
  • Wim Müller


The enzyme system from Clostridium sticklandii catalyzing the NADH-dependent reduction of d-proline was co-purified by chromatography on DEAE-cellulose at pH 8.2 and ammonium sulfate fractionation, and resolved into fractions containing three different protein components, NADH dehydrogenase, d-proline reductase and a third protein factor, by chromatography on DEAE-cellulose at pH 7.0. Upon recombination of the fractions containing the three different protein components, the NADH-dependent reduction of d-proline was successfully reconstituted. The NADH dehydrogenase fractions oxidized NADH in the presence of artificial electron acceptors, and were inhibited by p-hydroxymercuriphenylsulfonate (50% at 80 nM). They contained 3–4 different enzyme bands as revealed by polyacrylamide-gel electropherograms stained with the NADH-dependent reduction of 2,3,5-triphenyltetrazolium chloride. d-Proline reduction was also coupled to a leuco-methylene blue-generating system containing d-glucose and glucose-oxidase (EC Circumstantial evidence indicated that, among the clostridial proteins, only d-proline reductase and the third protein factor were needed for this reaction.

Key words

Clostridium sticklandii Stickland reaction Amino acid fermentation Two-substrate fermentation d-Proline reduction NADH dehydrogenase Leuco-methylene blue oxidation Electron transfer 

Non-standard abbreviations


2,3,5-triphenyltetrazolium chloride


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  1. Abeles, R. H.: d-Proline reductase (Clostridium sticklandii). In: Methods in Enzymology, Vol. 17 (H. Tabor, C. W. Tabor, eds.) pp. 317–321. New York and London: Academic Press 1971Google Scholar
  2. Barker, H. A.: Fermentation of nitrogenous organic compounds. In: The bacteria, Vol. 2 (I. C. Gunsalus, R. Y. Stanier, eds.), pp. 151–188. New York: Academic Press 1961Google Scholar
  3. Bentley, R.: Glucose oxidase. In: The enzymes, Vol. 7 (P. D. Boyer, H. A. Lardy, K. Myrbäck, eds.), pp. 567–586. New York: Academic Press 1963Google Scholar
  4. Boll, M.: Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum. Arch. Mikrobiol. 62, 94–110 (1968)Google Scholar
  5. Cone, J. E., del Rio, R. M., Stadtman, T. C.: Clostridial glycine reductase complex. Purification and characterization of the selenoprotein component. J. Biol. Chem. 252, 5337–5344 (1977)Google Scholar
  6. Gornall, A. G., Bardawill, C. J., David, M. M.: Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751–766 (1949)Google Scholar
  7. Mamelak, R., Quastel, J. H.: Amino acid interactions in strict anaerobes (Clostridium sporogenes). Biochim. Biophys. Acta 12, 103–120 (1953)Google Scholar
  8. Maurer, H. R.: Disk-Elektrophorese. Theorie und Praxis der diskontinuierlichen Polyacrylamidgel-Elektrophorese. Berlin: De Gruyter 1968Google Scholar
  9. Minakami, S., Ringler, R. L., Singer, T. P.: Studies on the respiratory chain-linked dihydrodiphosphopyridine nucleotide dehydrogenase. I. Assay of the enzyme in pariculate and soluble preparations. J. Biol. Chem. 237, 569–575 (1962)Google Scholar
  10. Nisman, B.: The Stickland reaction. Bacteriol. Rev. 18, 16–42 (1954)Google Scholar
  11. Ray, A., Reynolds, J. A., Polet, H., Steinhardt, J.: Binding of large organic anions and neutral molecules by native bovine serum albumin. Biochemistry 5, 2606–2616 (1966)Google Scholar
  12. Schwartz, A. C.: Enzymes involved in NADH-linked proline reduction in Clostridium sticklandii. Abstr. Commun. Meet. Fed. Europ. Biochem. Soc. 9, 490, Nr. f14.1 (1974)Google Scholar
  13. Schwartz, A. C., Schäfer, R.: New amino acids, and heterocyclic compounds participating in the Stickland reaction of Clostridium sticklandii. Arch. Mikrobiol. 93, 267–276 (1973)Google Scholar
  14. Seto, B.: Electron transport proteins associated with proline fermentation in Clostridium sticklandii. Fed. Proc. 37, 1521, abstract No. 1386 (1978)Google Scholar
  15. Seto, B., Stadtman, T. C.: Purification and properties of proline reductase from Clostridium sticklandii. J. Biol. Chem. 251, 2435–2439 (1976)Google Scholar
  16. Stadtman, T. C.: Studies on the enzymic reduction of amino acids: A proline reductase of an amino acid-fermenting Clostridium, strain HF. Biochem. J. 62, 614–621 (1956)Google Scholar
  17. Stadtman, T. C.: Studies on the enzymic reduction of amino acids. V. Coupling of a DPNH-generating system to glycine reduction. Arch. Biochem. Biophys. 99, 36–44 (1962)Google Scholar
  18. Stadtman, T. C.: Electron transport proteins of Clostridium sticklandii. In: Non-heme iron proteins. Their role in energy conversion (San Pietro, A., ed.), pp. 439–445. Yellow Springs, Ohio: Antioch Press 1965Google Scholar
  19. Stadtman, T. C.: Glycine reduction to acetate and ammonia: Identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system. Arch. Biochem. Biophys. 113, 9–19 (1966)Google Scholar
  20. Stadtman, T. C., Elliott, P.: Studies on the enzymic reduction of amino acids. II. Purification and properties of a d-proline reductase and a proline racemase from Clostridium sticklandii. J. Biol. Chem. 228, 983–997 (1957)Google Scholar
  21. Stein, A. M., Stein, J. H.: Studies on the Straub diaphorase. I. Isolation of multiple forms. Biochemistry 4, 1491–1500 (1965)Google Scholar
  22. Stickland, L. H.: The chemical reactions by which C. sporogenes obtains its energy. Biochem. J. 28, 1746–1759 (1934)Google Scholar
  23. Stickland, L. H.: Studies in the metabolism of the strict anaerobes (genus Clostridium). III. The oxidation of alanine by C. sporogenes. Biochem. J. 29, 889–896 (1935)Google Scholar
  24. Turner, D. C., Stadtman, T. C.: Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein. Arch. Biochem. Biophys. 154, 366–381 (1973)Google Scholar
  25. Warburg, O., Christian, W.: Isolierung und Kristallisation des Gärungsferments Enolase. Biochem. Z. 310, 384–421 (1941)Google Scholar

Copyright information

© Springer-Verlag 1979

Authors and Affiliations

  • Arnold C. Schwartz
    • 1
  • Wim Müller
    • 1
  1. 1.Botanisches Institut der Universität BonnBonn 1Germany

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