Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum |
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Authors: | L Dudley Eirich Roberts S Dugger |
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Institution: | Department of Biology, Portland State University, P.O. Box 751, Portland, OR 97207, U.S.A. |
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Abstract: | The F420-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F420 as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent Km of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH2 nor FADH2 could serve as electron donors; NAD was not utilized as electron acceptor. |
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Keywords: | NADP reductase Coenzyme F420 (Methanobacterium) |
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