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Genetic regulation of the utilisation of nitrate and ammonium in Aspergillus nidulans
Başlık:
Genetic regulation of the utilisation of nitrate and ammonium in Aspergillus nidulans
Yazar:
Dunn-Coleman, Nigel Stuart, author.
ISBN:
9780438060579
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (293 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: J. A. Pateman.
Özet:
The purpose of this study was to extend our knowled.ge of the regulation of enzyme activity in eukaryotic cells. For this purpose, the fungus Aspergillus nidulans, a simple eukaryote, was used. Five enzymes were studied, nitrate reductase, Hexokinase, Phosphoglucomutase, NADP-GDH and NAD-GDH. The latter four enzymes play an important role in the utilisation of glucose and glutamate. The effect of the nitrogen source and the carbon source on the regulation of these enzymes activity was examined. The reduction of nitrate to nitrite by nitrate reductase is the first step in the incorporation of inorganic nitrogen to form protein. Nitrate reductase in A. nidulans is an NADPH-linked enzyme and to reduce nitrate via nitrite to ammonia, much energy is used in the form of NADPH. When nitrate induced mycelium was transferred to various nitrogen and carbon treatments, there was a rapid inactivation of nitrate reductase activity, concurrent with a rapid decrease in the levels of the enzyme cofactor NADPH. In vivo studies of nitrate reductase regulation indicate that high levels of in vivo nitrate reductase activity can only be maintained when there is sufficient NADPH. In vivo studies of nitrate reductase also indicate that the level of nitrate reductase activity was dependent upon the concentration of NADPH. Cellfree extracts rapidly lose nitrate reductase activity when incubated. NADPH, but not nitrate, can prevent this loss of activity. This, loss of in vitro nitrate reductase activity was fully reversible. Cellfree extracts which had lost their nitrate reductase activity were fully reactivated upon the addition of NADPH. Mutants with impaired NADPH generation lost their in vitro nitrate reductase activity more rapidly than the wild type. The action of NADPH can be mimiced by the reducing agents, cysteine, and to a more limited extent Cleland's reagent, These results indicate that it is the redox state of NADPH and not any unique property of this pyridine nucleotide which is responsible for maintaining in vitro nitrate reductase activity. This activation/inactivation mechanism would provide a rapid means by which the nitrate reductase could be regulated and enable the cell to conserve NADPH. Hexokinase regulation was studied. Appreciable levels of Hexokinase activity were found with all carbon sources tested. Varying the nitrogen source did not affect the level of Hexokinase activity. One mutant, amrAl, has lowered Hexokinase activity and poor growth with a variety of carbon sources which may be the result of this, Phosphoglucomutase (PGlum) activity unlike Hexokinase does vary with both carbon and nitrogen source. Mycelium grown with ammonium has higher levels of PGlum activity than when mycelium is grown with nitrate, where the PGlum levels are lower. Low levels of PGlum would allow the glucose-6-phosphate genisrated by Hexokinase to be used to generate NADPH by the PPPenzyme G6PDH. Non-inducible mutants for the nitrate reductase regulatory gene nirA have high levels of PGlum when grown with nitrate. This indicates that the nirA gene may play a role in regulating the level of PGlum activity to allow more NADPH to be generated when mycelium is grown with nitrate. Ammonia derepressed mutants have nitrate reductase activity when grown with ammonium + nitrate. These mutants were found to have altered levels of NADPH generating enzymes and PGlum compared with the wild type when grown with ammonium + nitrate. These results indicate that ammonia derepression of nitrate reductase may be a result of high levels of NADPH generating ability in these mutants. The possibility that the level of NADPH in the cell may affect the synthesis of nitrate reductase was examined. Mutants with impaired NADPH generation were isolated and were found to have no detectable nitrate reductase activity. This indicates that the level of nitrate reductase cofactor, NADPH, can affect the synthesis of nitrate reductase. The regulation of NADP-GDH and NAD-GDH were studied, NADP-GDH activity was rapidly lost when the carbon source was removed. This inactivation of NADP-GDH was prevented by the protein synthesis inhibitor cycloheximide, Transfer of mycelium from conditions of carbon limitation to carbon sufficiency resulted in an increase in NADP-GDH activity even in the presence of cycloheximide. This result and immunological evidence indicate that under non-inducing conditions NADP-GDH is inactive, NAD-GDH activity was found to be highest under conditions of carbon limitation and lowest under conditions of carbon sufficiency. Immunological evidence indicates that the increase of NAD-GDH activity when mycelium was carbon limited was the result of de novo protein synthesis and not the activation of an inactive NAD-GDH.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684826.1) | 684826-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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