RESUMEN
The kinetics of NAD reduction by hydrogen, catalyzed by soluble hydrogenase from the hydrogen bacterium A. eutrophus Z-1 within a wide range of NAD substrate concentrations and pH were studied under aerobic and anaerobic conditions. The autocatalytic type of the reaction (with an induction period) and positive kinetic cooperativity with respect to NAD substrate at pH values greater than 8.0 were observed. A steady hydrogen release in a two-enzyme system involving hydrogenase, formiate dehydrogenase, formiate and NAD was demonstrated. A multistep pattern of the reaction mechanism of NAD reduction allowing to explain the autocatalytic type of NAD reduction by hydrogen as well as insensitivity of the reaction to air oxygen were proposed. Possible types of regulation of the soluble hydrogenase activity in the cell are discussed.
Asunto(s)
Alcaligenes/enzimología , NAD/metabolismo , Oxidorreductasas/metabolismo , Aerobiosis , Anaerobiosis , Concentración de Iones de Hidrógeno , Cinética , Oxidación-ReducciónRESUMEN
Soluble hydrogenase was isolated from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-1 and purified to electrophoretical homogeneity. The purification procedure included fractionation by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose and gelfiltration through Ultragel AcA-34. The resulting preparation had a specific activity of 25 mkmoles H2.min-1.mg of protein as measured by the rate of hydrogen evolution from sodium dithionite-reduced methyl viologen. The enzyme has a molecular weight of 200,000 and is made up of two subunits with mol. weights of 30,000 and two subunits with mol. weights of 65,000. The effects of pH, oxidants and reducers, as well as aerobic and anaerobic conditions on the hydrogenase preparations inactivation kinetics in intact cells and in a highly purified state were studied. The kinetic data suggest a possible existence of two enzyme forms differing in their activities and stabilities to denaturating influences.
Asunto(s)
Alcaligenes/enzimología , Oxidorreductasas/aislamiento & purificación , Aire , Argón , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Hidrógeno , Concentración de Iones de Hidrógeno , Isoenzimas/aislamiento & purificación , CinéticaRESUMEN
Homogenous yeast (Endomycopsis sp. 20-9) glucoamylase was isolated from cultural medium. The homogeneity of the enzyme preparation is demonstrated by means of polyacrylamide gel disc electrophoresis, isofocusing and ultracentrifugation. Amino acid composition, molecular weight (53 000), sedimentation constant (4.3S) and isoelectric point (pI 3.80-3.82) of the enzyme are determined. Glucoamylase is found to be glucoprotein.