Journal Article

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures

Conkling, M.A, Pharr, D.M, Willamson, J.D, Stoop, J.M.H. (North Carolina State University, Raleigh, NC.)

1995

Overview

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation of the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contributes to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 micromoles h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elusion. Sodium dodecyl sulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic 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

• Publisher: American Society of Plant Physiologists
• Subject: ACTIVIDAD ENZIMATICA / ACTIVITE ENZYMATIQUE / Amino Acid Sequence / ANTICORPS / ANTICUERPOS / APIUM GRAVEOLENS / Biochemistry and Enzymology / Biological and medical sciences / Celery / cell suspension culture / Cells, Cultured / Chromatography / Chromatography, Ion Exchange / CLONACION / CLONAGE / COENZIMAS / COENZYME / COMPOSICION QUIMICA / COMPOSITION CHIMIQUE / CULTIVO DE CELULAS / CULTURE DE CELLULE / cytology / Dehydrogenases / enzyme activity / Enzymes / enzymology / Fractionation / Fundamental and applied biological sciences. Psychology / Gels / GENE / GENES / genetic engineering / genetics / GENIE GENETIQUE / INGENIERIA GENETICA / isolation & purification / MANITOL / MANNITOL / Metabolism / Molecular Sequence Data / Mops / NAD / NAD (coenzyme) / NAD - metabolism / NUCLEOTIDE / NUCLEOTIDOS / oxidoreductases / OXIDORREDUCTASAS / OXYDOREDUCTASE / Parsley / Plant physiology and development / Plants / polyclonal antibodies / protein composition / PROTEINAS / PROTEINE / Proteins / PURIFICACION / PURIFICATION / Sugar Alcohol Dehydrogenases / Sugar Alcohol Dehydrogenases - genetics / Sugar Alcohol Dehydrogenases - isolation & purification / Vegetables / Vegetables - cytology / Vegetables - enzymology