Abstract
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 on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute 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 mumol 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 elution. Sodium dodecylsulfate 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 immuno-reactive 40-kD protein.
Full Text
The Full Text of this article is available as a PDF (1.7 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Rumpho M. E., Edwards G. E., Loescher W. H. A pathway for photosynthetic carbon flow to mannitol in celery leaves : activity and localization of key enzymes. Plant Physiol. 1983 Dec;73(4):869–873. doi: 10.1104/pp.73.4.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stoop J. M., Pharr D. M. Partial purification and characterization of mannitol: mannose 1-oxidoreductase from celeriac (Apium graveolens var. rapaceum) roots. Arch Biochem Biophys. 1992 Nov 1;298(2):612–619. doi: 10.1016/0003-9861(92)90456-7. [DOI] [PubMed] [Google Scholar]
- Stoop JMH., Pharr D. M. Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures. Plant Physiol. 1993 Nov;103(3):1001–1008. doi: 10.1104/pp.103.3.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tarczynski M. C., Jensen R. G., Bohnert H. J. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science. 1993 Jan 22;259(5094):508–510. doi: 10.1126/science.259.5094.508. [DOI] [PubMed] [Google Scholar]
- Ueng S. T., Hartanowicz P., Lewandoski C., Keller J., Holick M., McGuinness E. T. D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions. Biochemistry. 1976 Apr 20;15(8):1743–1749. doi: 10.1021/bi00653a023. [DOI] [PubMed] [Google Scholar]