博士 / 國防醫學院 / 生命科學研究所 / 91 / ABSTRACT
Synechococcus sp. strain RF-1 is a unicellular freshwater cyanobacterium that fixes N2 aerobically and exhibits a circadian rhythm for nitrogenase activity under a light/dark regimen. Synechococcus sp. strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth. Nitrate uptake in Synechococcus sp. strain RF-1 cells was experimentally induced by nitrate or nitrite and the induction repressed by ammonium. Nitrate uptake was tightly associated with the CO2 fixation in Synechococcus sp. strain RF-1, and its feedback inhibition was eliminated as glutamine synthetase (GS) inhibited by MSX (L-methionine-D,L-sulfoximine). In the presence of MSX, nitrate uptake was significantly enhanced by citrate addition. We provided the in vivo evidence to suggest that nitrate uptake in Synechococcus sp. strain RF-1 cells is tightly regulated by internal C/N status of cells. In contrast, the in situ nitrate reductase (NR) activity was differentially regulated from nitrate uptake in Synechococcus sp. strain RF-1, whose NR activity was not inhibited by ammonium and only slightly enhanced by nitrate. The in situ NR of Synechococcus sp. strain RF-1 exhibited biphasic kinetics for nitrate was derived from the single narB gene that was verified by gene expressed in heterologous host of E coli. Both the high- and low-affinity NR activities of recombinant NarB were not distinguishable by the heat denaturing treatments and showed the same thermostability. Furthermore, monomer NarB catalyzed the both high- and low-affinity NR activities on the alkaline native gel-NR activity assay. When poly-histidine tagged at N-terminal of NarB or NaN3 treatment, the recombinant NR was shifted from biphasic to monophasic kinetics with single Km for nitrate indicated that functional importance of the NarB terminal structure in NR kinetics and the enzyme transition was interfered by NaN3 treatment.
NR Synechococcus sp. strain PCC 7942 had been published with single Km for nitrate. However, we found the NarB of Synechococcus sp. strain PCC 7942 actually also exhibited the biphasic kinetics. In the present study, we provide several lines of evidence to indicate that the biphasic kinetics of NR for nitrate is an intrinsic nature of the NarB-type cyanobacterial NR.
| Identifer | oai:union.ndltd.org:TW/091NDMC0105012 |
| Date | January 2003 |
| Creators | Tung-Hei Wang, 王東海 |
| Contributors | Yuh-Jang Shieh, 謝昱暲 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 72 |
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