Thesis (Ph. D.)--University of Wyoming, 2005. / Title from PDF title page (viewed on Nov. 7, 2007). Includes bibliographical references (p. 117-135).

Inaug.-diss.--Leipzig. / Lebenslauf.

Inaug.-diss.--Leipzig. / Vita. "Litterartur-verzeichnis": p. [43].

Inaug.-diss.--Breslau. / Lebenslauf. "Literstur-verzeichnis": p. [57]-58.

Inaug.-diss.--Rostock.

Inaug.-diss.--Giessen. / Lebenslauf. "Literatur": p. [4].

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

A soluble DPNH-nitrate reductase (NRase) of the sugar beet has been purified and characterized. The occurrence of NO₂⁻ as an end product and the insensitivity of the enzyme to oxygen indicate that the sugar beet NRase is of the nitrate assimilation type. The enzyme was not associated with any cell particle and all NRase activity present in the homogenate of sugar beet leaves was recovered in the 20,000 x g supernatant. A sixtyfold purification was accomplished by ammonium sulfate precipitation followed by adsorption on calcium phosphate gel. At room temperatures and higher the enzyme was heat labile, but was relatively stable at -15°C. Dialysis at 4° C. did not result in an appreciable loss of activity. The optimum pH was 7.0. The NRase was sensitive to heavy metal inhibitors but it was not possible to show that Mo was the specific prosthetic metal. It was demonstrated, however, that chemically reduced Mo could serve as an electron donor. Thus Mo may be a cofactor for the enzyme. The reversal of p-chloromercuribenzoate inhibition by the sulfhydryl reagents glutathione and cysteine, coupled with strong inhibition by iodoacetate and cupric sulfate indicated the sulfhydryl nature of the enzyme. The partially purified NRase was stimulated to a considerably greater degree by FAD than by FMN. Rf values and co-chromatography in different solvents showed that a substance liberated from the enzyme preparation by acid and heat was not riboflavin or FMN but very probably was FAD. It is suggested that, in common with other assimilatory NRases of higher plants, the flavin nucleotide prosthetic group of sugar beet NRase is FAD. The presence of two NRases in the sugar beet was indicated by the fact that the crude "enzyme" was stimulated to the same extent by the addition of DPNH or TPNH, that the ratio of activities resulting from the addition of the two pyridine nucleotides changed with the degree of purity of the enzyme, and that the enzyme finally obtained by calcium phosphate gel adsorption and elution was DPNH-specific. That purification was not complete was shown by the presence of DPNH-quinone reductase and DPNH-cytochrome c reductase activity in the NRase preparation. A low NRase activity and a high nitrate content were measured in sugar beet leaves during growth in darkness. The reverse occurred in light. It is suggested that the diurnal variation in NRase activity may be the result of the fall of leaf tissue pH during darkness and its rise to approximate the enzyme's optimum pH in light. The possible participation of the NRase in a flavin nucleotide-catalyzed enzymatic photoreduction of nitrate was indicated by the coupling of photoreduction of FAD with the reduction of nitrate by NRase. / Science, Faculty of / Botany, Department of / Graduate

Sugar beet seed, S.K.E.-R-11, was obtained from the B.C. Sugar Co. Ltd., Vancouver, B.C. and germinated in flats in a greenhouse on January 29, 1954. The beet plants were transplanted to a fertilized field on May 2, 1954. A randomized lot design was chosen in order to reduce error caused by soil differences, moisture variations, and pH etc. Thirty groups of 10 beets per group were selected from the randomized lot. The leaves of 6 groups were frozen with dry ice and the other groups were defoliated, decrowned continuously defoliated, or used as controls. The regrowth on the continuously defoliated beets was removed every two days following initial defoliation. Defoliation was effected by slicing off the leaves one-quarter inch above the crown. Decrowning was done by cutting the beet root transversely just beneath the outer ring of meristematic buds. The: defoliated beets were used to serve as a parallel to the destruction of leaves by freezing. The continuously defoliated beets were a check on the defoliated beets, where it was considered that photosynthesis in the new regrowth leaves would partially offset a large sugar loss in the root. Two experiments were completed. The first experiment and treatment began on August 16, 1954; the second on October 13, 1954. Harvest times were at the 1, 4, 8, 11, 16 and 20 day intervals following Aug. 16, and at the 1, 4, 8, 12 and 15 day intervals following October 13. Enzyme activity only was determined in the second experiment. 'The fresh leaf weights of the defoliated and control beets were recorded and later compared with leaf regrowth weights and sugar content. The beets were harvested in groups of 10 beets all treated in one specific manner. Ten beets of each group were removed from the soil and each beet sliced diagonally across the centre region. The sections were washed in water and pulped in a meat grinder giving approximately 2000 grams of pulp from 10 sections. Three hundred grams of pulp was used in dry weight -determination. Forty grams of fresh pulp from each group was blended for 2 minutes with 100 ml. of distilled ice water in a Waring blendor. The solution was filtered through broadcloth and used in enzyme activity measurements. In the second experiment, 47 grams of pulp was blended with 100 ml. of distilled ice water for 2 minutes. The crown portion of the root was used in the estimation of invertase activity. A check on the sampling method showed that the 40-gram aliquot of pulp used for enzyme determination represented the sample. Sucrose percent and phosphatase activity were used as the basis of this test. The fresh pulp was analyzed for sucrose, invert sugars, dry weight, catalase, phosphorylase, beta-amylase and invertase enzyme activities. The dried pulp was ground to 40-mesh and analyzed for total nitrogen, sucrose and invert sugars. Insoluble nitrogen and starch-dextrins were determined in ethanol extracted pulp. Duplicate determinations were made on each sample. Percentages are based on both dry and fresh weights and given as T/C values. Phosphorylase, phosphatase, catalase, beta-amylase, invertase were measured. Sucrose, invert sugars, starch-dextrin3, and total and insoluble nitrogen were also determined. The.highest amount of leaf regrowth occurred 4-17 days after freezing. The results indicated no relation between leaf weights and sucrose content nor between root weight and sugar content in mature beets. The percent dry weight decreased in all treated beets from the 1st to the 20th days after treatment. This decrease is likely a result of sucrose loss and an increased hydration in the beet root. Sucrose percent based on dry and fresh weight generally decreased following all- treatments. A positive correlation between percent sugar loss and leaf regrowth is suggested. There was an increase in the amount of reducing sugars after foliar loss. The suggestion has been made that the monosaccharide sugars are utilized almost immediately in leaf regrowth or in (increased respiration in the beet crown. The percent, of starch-dextrins tended to decrease in the treated beets but this is most likely not significant. The decrease in percent of total carbohydrates found follows the fact that sucrose disappears. Total carbohydrate estimations seem to provide a reasonable basis for determining the amount of sucrose loss. Total and soluble nitrogen values decreased to the 8th day after treatment and increased after this time. Insoluble nitrogen results were generally inconclusive. The results suggested a translocation of soluble nitrogenous compounds to the beet crown where active growth was occurring. The apparent activity of phosphorylase decreased with time in all treatments. Starch phosphorylase in sugar beet root likely has a minor role in total carbohydrate metabolism of the tissue. Phosphatase activity decreased to -the 11th day in every treatment except decrowned. The reason for a lower apparent phosphatase activity in treated beets in this experiment is not known. It may be associated with an increase respiratory rate. There were no significant changes in beta-amylase activity and no correlation could be found between starch-dextrin content and amylase activity. Catalase activity based on monomolecular values, decreased with time after treatment. A decrease in catalase activity might be expected in the mature, cells of the root as the respiration rate decreases with age. A correlation between invertase activity and sucrose loss was indicated in the frozen and decrowned beets but not in the defoliated beets. From the results of this experiment it seems unlikely that invertase is alone responsible for a sucrose decrease. The results found in this experiment were largely negative. / Science, Faculty of / Botany, Department of / Graduate

Metabolic inhibitors and growth regulators were used in an attempt to control the growth of sugar beet plants at the time of "ripening" of the roots. Maleic hydrazide (MH), pyrocatechol (PC), and vanadium sulphate (VS) were found to be most effective in controlling growth regardless of the age of the plants. The solutions containing MH, PC, or VS were applied to the foliage of 4.5-month-old plants and the effects on leaf expansion and content of sucrose, reducing sugars, nitrite, nitrate, ammonia, amino acid, protein and total nitrogen were determined 7, 14, and 21 days after treatment. The rate of photosynthesis and respiration and the activity of nitrate reductase, transaminase, invertase, adenosine triphosphatase (ATP-ase), glucose-1-, glucose-6-, fructose-6-phosphatase, uridine diphosphate glucose pyrophosphorylase (UDPG-pyrophosphorylase), sucrose synthetase and sucrose phosphate synthetase was measured. Compared with untreated plants, with few exceptions, all treatments affected the growth; the chemical compositions the rate of photosynthesis and respiration, and the activities of enzymes measured, in a similar manner. Growth of the plants was determined by measuring the leaf area. MH, PC, and VS significantly inhibited growth of leaves under both "summer" and "fall" conditions. In the treated plants, the percentage reducing sugars, based on fresh weight of the root, decreased and percentage sucrose increased steadily. Application of MH, PC, and VS resulted in a significant decrease in nitrite and an increase in nitrate content of roots. Ammonium nitrogen of the plants treated with MH was more than that of the untreated plants on the 7th, 14th, and 21st day after treatment. Plants reacted with PC and VS had a lower ammonium content on the 7th and the 14th day but more on the 21st day. The soluble amino acid content of the roots of MH-treated plants was higher than that of the controls. PC-treated plants had a lower amino acid content on the 7th day but a higher content on the 14th and 21st day. VS caused a reduction in amino acid content of the roots on all dates of harvest. The rate of photosynthesis was measured by infrared technique. MH and VS caused a stimulation in the rate of net C0₂ assimilation, however, PC inhibited the rate of net C0₂ assimilation on the 7th day after treatment. The rate of respiration of the storage roots, measured by the Warburg technique, was lower than that of the control plants in the case of MH-and VS-treated plants and it was higher in the PC-treated plants. The results indicated that the application of MH, PC, and VS caused significant reduction in the activity of nitrate reductase, transaminase, inver-tase, ATP-ase, glucose-1-, glucose-6-, and fructose-6-phosphatase. These treatments also resulted in the stimulation of the activity of UDPG-pyrophos-phorylase, sucrose synthetase and sucrose phosphate synthetase. The inhibition of growth by MH, PC, and VS is discussed on the bases of the reductions in the activities of invertase, nitrate reductase, and transaminase. The increase in sucrose content of the roots is explained on the bases of low invertase and high sucrose synthetase and sucrose phosphate synthetase activities in the treated plants. The possible participation of the phosphatases in the regulation of sucrose biosynthesis is indicated by the negative correlations between the activities of phosphatases and sucrose phosphate synthetase. / Science, Faculty of / Botany, Department of / Graduate