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Title: Studies of the biosynthesis and properties of nebularine
Author: Konuk, M.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1993
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Although Lepista (Clitocybe) nebularis has been examined at the beginning of this present study, its slow rate of nebularine production necessitated finding an alternative nebularine producer. Streptomyces yokosukanensis was examined and shown to be suitable for a biosynthetic study. Using Streptomyces yokosukanensis adenosine was shown to be the immediate precursor of nebularine (9-β-D-ribofuranosyl purine). Inosine and guanosine were not active in this respect. An HPLC method was developed for nebularine and it was found to be more sensitive, accurate and quicker than extraction, purification and spectrophotometry. Using the HPLC method, the nebularine production of both Lepista nebularis and Streptomyces yokosukanensis was examined. With [8-^14C]adenosine, purine and nebularine 5'-phosphate were detected as metabolites in cell extracts of Lepista nebularis and of Streptomyces yokisukanensis. Metabolism of [8-^14C]adenosine to other purines was examined in Streptomyces yokosukanensis. The nebularine-forming enzyme was extracted and partially purified by ammonium sulphate fractionation during which it remained in the 100% saturated supernatant. Further purification was achieved with gel filtration chromatography of a Sephacryl S-200 column and with hydrophobic affinity chromatography on BrCN activated Sepharose 4B. The M_r of the enzyme was estimated to be around 10,000. It had no detectable coenzyme requirement. Similarly, a range of metal ions were without observable effect. Among the range of purine ribosides, nebularine was found to inhibit the nebularine-forming enzyme activity. The enzyme, which has a pH optimum at 7.0, catalyses the removal of the 6-amino group from adenosine as hydroxylamine. This was demonstrated chemically and using ^15N-mass spectrometry. Nebularine was found to have very strong inhibitory effect on the growth of wheat, lettuce, spinach and carrot seedlings. It is a strong antagonist of cytokinin activity as demonstrated by both the Amaranthus and the leaf senescence bioassays. It was also found to be a non-competitive inhibitor of xanthine oxidase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available