Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile |
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Authors: | Takamoto Suzuki Yoshiharu Maruyama Michinori Nakamura |
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Institution: | Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan |
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Abstract: | The Tritium (T) uptake method for detecting hydrogenase (Hase) was applied to measure the Hase activity of aerobic nitrogen-fixing bacterium Azotobacter agile. The cell-free extract of this bacterium contains the ATP-stimulated T-uptake activity, and this activity was separated from the nitrogenase activity. In the supernatant obtained by centrifugation at 20,000 × g for 30 min, this ATP-stimulated T-uptake activity existed mainly in large molecular weight fraction and was distributed to precipitate at 184,000 × g for 1 hr. After this ultra-centrifugation, the distribution patterns of methylene blue (MB) reduction and T-uptake activities were significantly different from each other, and MB reduction activity remained much more in the supernatant. The Hase activity detected by both T-uptake and MB reduction was mainly in the particle fraction precipitated at 20,000 × g for 30 min from the cell-free extract. When the activities of the praticle fraction were solubilized with Triton X–100, the ATP-stimulated T-uptake activity was effectively solubilized. These results imply that the cell-free extract of Azotobacter agile contained some different kinds of hydrogenases which catalyzed MB reduction, T-uptake and ATP-stimulated T-uptake activities at different intensities from each other. |
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