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

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.Non-standard abbreviations TTC 2,3,5-triphenyltetrazolium chloride