Phosphomannose isomerase in Nicotiana tabacum L. NT1 and Apium graveolens var. dulce L. cell suspension cultures.

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Title: Phosphomannose isomerase in Nicotiana tabacum L. NT1 and Apium graveolens var. dulce L. cell suspension cultures.
Author: Barb, Adam Wesley
Advisors: Dr. John D. Williamson, Committee Co-Chair
Dr. Yuri Yamamoto, Committee Member
Dr. D. Mason Pharr, Committee Co-Chair
Abstract: Phosphomannose isomerase (PMI) [E.C.] is a key enzyme required for Man metabolism in plants, and PMI activity is important in both mannitol metabolizing and mannitol non-metabolizing plants. In this manuscript, two studies are presented; one that describes PMI activity in a mannitol non-metabolizing plant, Nicotiana tabacum L. NT1 cell suspension cultures, and one that describes the partial purification of PMI from a mannitol metabolizing plant, Apium graveolens var. dulce. Wild type cultures of NT1 cells grew slowly with Man as the sole carbohydrate source, doubling every 158 h, whereas NT1 cells doubled every 26 h when grown on Glc as the sole carbohydrate source. A mutant line of NT1 cells was selected from the wild type culture by repeated subculture into fresh Man medium, and after three months, exhibited a doubling time of 38 h on Man as the sole carbohydrate source. Additionally, this mutant culture exhibited a five-fold higher PMI activity level than the wild type culture from which it was selected. Interestingly, we noted that this mutant culture had a decreased growth rate on Glc, with a culture doubling time of 31 hours, and was found to have more than a 50% reduction in hexokinase (HK) [] activity. In the second study, A. graveolens cell suspension cultures were shown to have high levels of PMI activity (200 μmol hr-1 g-1 fresh weight) and provided an excellent starting material for the purification of PMI. PMI from Glc-grown A. graveolens cultures was purified 23.5-fold with a 6% total recovery of PMI activity, using ammonium sulfate precipitation, gel filtration chromatography, and ion exchange chromatography. Low recovery of PMI activity was presumably because this activity was not stable. Although protease inhibitors in the extraction buffer resulted in the recovery of nearly twice the amount of nonspecific protein and PMI activity, neither protease inhibitors nor the addition of zinc to the enzyme solution aided in the maintenance or recovery of PMI activity in purification steps past ammonium sulfate precipitation. Additionally, different affinity chromatography resins did not bind PMI activity, but might still serve as a useful tool later in the purification of the enzyme.
Date: 2002-07-03
Degree: MS
Discipline: Horticultural Science

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