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
Article

Abstract

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 1.1.3.4). 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

TTC

2,3,5-triphenyltetrazolium chloride

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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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