| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1987: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
In this research, we investigated energy metabolism and typical CO_2 fixation reaction in Hydrogenobacter thermophilus.
1. Energy metabolism in Hydrogenobacter thermophilus. By using cell lysate and membrane fraction, it was shown that cytochrome c_<552> was reduced by hydrogenase reaction. So, it was demonstrated that cytochrome c_<552> is a natural electron acceptor for the membrane-bound hydrogenase. In order to confirm the result, membrane-bound hydrogenase and cytochrome c_<552> were purified. The membrane-bound hydrogenase was purified by the following steps (1)Solubilization, (2)Hydrophobic chromatography, (3)Adsorption chromatography, and (4)Ion-exchange chromatography. Cytochrome c_<552> was purified by the following steps (1)Ion-exchange chromatography, and (2)Affinity chromatography. In vitro experiment showed that the purified hydrogenase reduces cytochrome c_<552> in the presence of detergents. Together with the fact that the terminal cytochrome in H. thermophilus is cytoch
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rome o, we clarified the electron transport system in H. thermophilus.
2. Typical CO_2 fixation reaction in H. thermophilus. H. thermophilus is known to fix CO_2 via a reductive carboxylic acid cycle. But, in two typical CO_2 fixation reactions (pyruvate synthase and -ketoglutarate synthase), the activities could not be measured in forward directions. To know what kind of reductant is involved in H. thermophilus, we tried to demonstrate the enzyme activity in forward direction. For that purpose, we obtained a partially purified puruvate synthase by ion-exchange chromatography and various reductants. To obtain a partially purified enzyme, the enzyme activity was assayed in reverse direction. As reductants, methylviologen, membrane extracts, and NAD(P)H were used. Unfortunately, under the various conditions we tried, we could not demonstrate the enzyme activity in forward direction. It is known that pyruvate synthase catalyzes isotope exchange reaction between NaH^<14>CO_3 and pyruvate. As the partially purified enzyme catalyzed this reaction, it became clear that pyruvate synthase really works in H. thermophilus. Less
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