Purification and partial characterization of proline dehydrogenase from Clostridium sporogenes.

A proline dehydrogenase which catalyzes the nicotinamide adenine dinucleotide (NAD) dependent oxidation of proline and the NADH-dependent reduction of delta 1-pyrroline 5-carboxylic acid (PCA) was purified from extracts of Clostridium sporogenes. Following purification, only one protein band was found on analytical polyacrylamide disc gels and on sodium dodecyl sulfate (SDS) - polyacrylamide disc gels. Sucrose density gradient centrifugation and SDS-gel electrophoresis indicated that the enzyme has a molecular weight of approximately 217 000 and consists of two subunits of equal size. During purification of proline dehydrogenase on hydroxylapatite the ratio of dehydrogenase activity to reductase activity decreases significantly, and a similar change in ratio was brought about by storage of partially purified enzyme preparations in low ionic strength buffers. Subsequent purification did not change the ratio. The dehydrogenase activity of proline dehydrogenase was inhibited by L-glutamate (Ki = 0.32 mM at pH 7.4 and Ki = 0.65 mM at ph 10.2). However, the reductase activity of the purified enzyme was not affected by 100 mM L-glutamate.