UBC Theses and Dissertations
Disphosphopyridine nucleotide-nitrate reductase in Beta vulgaris L. Yang, Kuan Jen
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.
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