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

Abstract

The F 420 -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 F 420 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 ~M for reduced F 420 , and an apparent Km of 4lfM 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. Thanks should also go to Mary Taylor for always being there to lend a hand or the benefit of her experience. Mary is also one of those special educators who always have time for their students.I must give special credit to Sanjay Khandekar, the Test Tube King, for painstakingly making sure the spectrophotometer tubes were never without a protective coat of rust, and for giving me se~eral useful sources for this thesis. Also, he's kind of funny. In systems where sulphur reduction to hydrogen sulfide is an important pathway, methanogenesis is retarded, suggesting that the two reactions are competit~ve [7].Methanogenic bacteria may be found in systems with widely varying conditions, cool to very hot, organically rich or poor, and high or low pressure. Some methanogens require only hydrogen and carbon dioxide, while others require additional nutrients. Those ecosystems containing mostly carbon dioxide of geochemical origin are known as the chemolithotrophic systems. They occur primarily in thermal springs and lakes where volcanic intrusion of some kind is present [7]. The methanogens present in these environments are often quite hardy, thermophilic, and obviously are efficient at obtaining energy and cell mass from compounds which are unmetabolizable to other organisms.The other type of methanogenic ecosystem contains large to moderate amounts of organic monomers and polymers of biogenic and/or synthetic origin. This type of environment occurs in the digestive tracts of various animals, especially in the ruminants, and in places where material is being biodegraded anaerobically, such as swamps, bogs, sediments, water-saturated soils, sewage sludge, liquid waste digestors, biogas installations, garbage dumps, and landfills. It is in some of these environments that the presence of methanogens is important, both as the final link in the anaerobic food chain, and as scavengers which help control the pH and partial pressure of hydrogen, factors which are extremely important for the complete dissimilation of organic material in anaerobic environments.In these systems, which are almost always mixed cultures of several methanogen...