PURIFICATION OF NAD-DEPENDENT MANNITOL DEHYDROGENASE FROM CELERY SUSPENSION-CULTURES

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitut es the first enzymatic step in the catabolism of mannitol in nonphotos ynthetic tissues of celery (Apium graveolens L.). Endogenous regulatio n of the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, c ontributes to stress tolerance of celery. The enzyme was purified to h omogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a speci fic activity of 365 mu mol h(-1) mg(-1) protein with a 37% yield of en zyme activity present in the crude extract. A highly efficient and sim ple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NA D-agarose affinity chromatography using NAD gradient elution. Sodium d odecyl sulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was de termined to be approximately 43 kD, indicating that the enzyme is a mo nomer. Polyclonal antibodies raised against the enzyme inhibited enzym atic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immunoreactive 40-kD protein.