NADH-dependent reduction of d-proline in Clostridium sticklandii. Reconstitution from three fractions containing NADH dehydrogenase, d-proline reductase, and a third protein factor Article DOI:
10.1007/BF00446821 Cite this article as: Schwartz, A.C. & Müller, W. Arch. Microbiol. (1979) 123: 203. doi:10.1007/BF00446821 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 18.104.22.168). 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
Abeles, R. H.:
-Proline reductase (
). In: Methods in Enzymology, Vol. 17 (H. Tabor, C. W. Tabor, eds.) pp. 317–321. New York and London: Academic Press 1971
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 1961
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 1963
Boll, M.: Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in
. Arch. Mikrobiol.
, 94–110 (1968)
Cone, J. E., del Rio, R. M., Stadtman, T. C.: Clostridial glycine reductase complex. Purification and characterization of the selenoprotein component. J. Biol. Chem.
, 5337–5344 (1977)
Gornall, A. G., Bardawill, C. J., David, M. M.: Determination of serum proteins by means of the biuret reaction. J. Biol. Chem.
, 751–766 (1949)
Mamelak, R., Quastel, J. H.: Amino acid interactions in strict anaerobes (
). Biochim. Biophys. Acta
, 103–120 (1953)
Maurer, H. R.: Disk-Elektrophorese. Theorie und Praxis der diskontinuierlichen Polyacrylamidgel-Elektrophorese. Berlin: De Gruyter 1968
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.
, 569–575 (1962)
Nisman, B.: The Stickland reaction. Bacteriol. Rev.
, 16–42 (1954)
Ray, A., Reynolds, J. A., Polet, H., Steinhardt, J.: Binding of large organic anions and neutral molecules by native bovine serum albumin. Biochemistry
, 2606–2616 (1966)
Schwartz, A. C.: Enzymes involved in NADH-linked proline reduction in
. Abstr. Commun. Meet. Fed. Europ. Biochem. Soc.
, 490, Nr. f14.1 (1974)
Schwartz, A. C., Schäfer, R.: New amino acids, and heterocyclic compounds participating in the Stickland reaction of
. Arch. Mikrobiol.
, 267–276 (1973)
Seto, B.: Electron transport proteins associated with proline fermentation in
. Fed. Proc.
, 1521, abstract No. 1386 (1978)
Seto, B., Stadtman, T. C.: Purification and properties of proline reductase from
. J. Biol. Chem.
, 2435–2439 (1976)
Stadtman, T. C.: Studies on the enzymic reduction of amino acids: A proline reductase of an amino acid-fermenting
, strain HF. Biochem. J.
, 614–621 (1956)
Stadtman, T. C.: Studies on the enzymic reduction of amino acids. V. Coupling of a DPNH-generating system to glycine reduction. Arch. Biochem. Biophys.
, 36–44 (1962)
Stadtman, T. C.: Electron transport proteins of
. In: Non-heme iron proteins. Their role in energy conversion (San Pietro, A., ed.), pp. 439–445. Yellow Springs, Ohio: Antioch Press 1965
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.
, 9–19 (1966)
Stadtman, T. C., Elliott, P.: Studies on the enzymic reduction of amino acids. II. Purification and properties of a
-proline reductase and a proline racemase from
. J. Biol. Chem.
, 983–997 (1957)
Stein, A. M., Stein, J. H.: Studies on the Straub diaphorase. I. Isolation of multiple forms. Biochemistry
, 1491–1500 (1965)
Stickland, L. H.: The chemical reactions by which
obtains its energy. Biochem. J.
, 1746–1759 (1934)
Stickland, L. H.: Studies in the metabolism of the strict anaerobes (genus
). III. The oxidation of alanine by
. Biochem. J.
, 889–896 (1935)
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.
, 366–381 (1973)
Warburg, O., Christian, W.: Isolierung und Kristallisation des Gärungsferments Enolase. Biochem. Z.
, 384–421 (1941)