Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica.
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.
Ewart
G D
GD
Department of Biochemistry, Faculty of Science, Australian National University, Canberra, ACT.
Smith
G D
GD
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
D006864:Hydrogenase