Partial purification and characterization of F₄₂₀-dependent NADP reductase from Methanobrevibacter smithii strain DE1

• Main Author: Sheridan, Scott D.
• Format: Others
• Published: PDXScholar 1985
• Subjects: Nicotinamide adenine dinucleotide phosphate; Methanobrevibacter smithii; Anaerobic bacteria; Biology; Enzymes and Coenzymes
• Online Access: https://pdxscholar.library.pdx.edu/open_access_etds/3523; https://pdxscholar.library.pdx.edu/cgi/viewcontent.cgi?article=4532&context=open_access_etds

The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.