In this paper the purification is reported of a protein which is able to catalyze both the proline oxidase and the pyrroline-5-carboxylic acid dehydrogenase activities necessary for the oxidation of proline to glutamic acid. The purification involves the preparation of a crude membrane pellet, detergent solubilization, ammonium sulfate fractionation and DEAE-chromatography. An essential pure preparation (>95% pure) is obtained after only a 52-fold purification, demonstrating that the protein is a major protein in cells fully induced for proline utilization. Both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activates co-purity throughout our purification. Velocity sedimentation of the purified protein demonstrates that both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities co-sediment. Early in the purification procedure two species of protein which have both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities were detected. The procedure purifies only the larger molecular weight species. The purified protein is a dimer composed of identical 132,000-dalton subunits. Analysis of mutants defective for proline utilization demonstrate that the bifunctional enzyme is the putA gene product.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1981|
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