Some properties of the nickel-containing hydrogenase of chemolithotrophically grown Rhizobium japonicum.
The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.
D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009532:Nickel / Q000032:analysis*; D012231:Rhizobium / Q000201:enzymology* / Q000254:growth & development
D009532:Nickel; D006859:Hydrogen; D006864:Hydrogenase