title: Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica.
abstract: Two soluble hydrogenase activities were separable from cell extracts of the cyanobacterium Anabaena cylindrica, one detectable by the tritium exchange assay, the other having a relatively low tritium exchange activity but catalyzing methyl viologen-dependent hydrogen formation. Their molecular weights, by gel filtration chromatography, were 42,000 and 100,000, respectively. The two hydrogenase activities were differentially inhibited. The methyl viologen-dependent activity has been purified to homogeneity from cells in which the enzyme was induced by gassing the growing cells with N2/H2/CO2 (95.7%/4%/0.3%, v/v/v). The procedure involved French pressure cell disruption of the cells, differential precipitation with ZnCl2, heat treatment (50 degrees C), and lyophilization of the heat-step supernatant. It was then subjected to DEAE-Sephacel chromatography, dye-ligand chromatography on Procion Red, and HPLC anion exchange on QMA-Accel. Polyacrylamide gel electrophoresis on both native and denaturing gels revealed two peptides with Mr's 42,000 and 50,000. The 42,000 protein alone catalyzed tritium exchange activity; both proteins appeared to be necessary for the methyl viologen activity. The native enzyme appears to be a readily dissociable dimer of two nonidentical subunits, one of which contains the hydrogen binding site and the other providing the ability to utilize electrons from a reductant for hydrogen formation.
authors.affiliation: Department of Biochemistry, Faculty of Science, Australian National University, Canberra, ACT.
authors.forename: G D
authors.initials: GD
authors.lastname: Ewart
authors.forename: G D
authors.initials: GD
authors.lastname: Smith
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D012995:Solubility
chemical_list: D006864:Hydrogenase