title: Characterization of extracellular Mn2+-oxidizing activity and isolation of an Mn2+-oxidizing protein from Leptothrix discophora SS-1.
abstract: Supernatant fluid from Leptothrix discophora SS-1 cultures possessed high Mn2+-ozidizing activity. Studies of temperature and pH optima, chemical inhibition, and protease sensitivity suggested that the activity may be enzymatic. Kinetic studies of unconcentrated supernatant fluid indicated an apparent Km of 7 microM Mn2+ in the 1 to 200 microM Mn2+ range. The greatest Vmax value observed was 1.4 nmol of Mn2+ oxidized min-1 micrograms of protein-1 in unconcentrated samples. When the supernatant fluid was concentrated on DEAE-cellulose and the activity was eluted with MgSO4, an Mn2+-oxidizing protein was detected in the concentrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mn2+-oxidizing protein appeared to have a molecular weight of 110,000 in 10% polyacrylamide gels and of 100,000 in 8% gels. Periodic acid-Schiff base staining of overloaded polyacrylamide gels showed that the DEAE-cellulose concentrate contained abundant high-molecular-weight polysaccharides; concurrent staining of the Mn2+-oxidizing band suggested that it too contained carbohydrate components. Isolation of the protein was achieved by subjecting the DEAE-cellulose concentrate to Sephacryl gel filtration in the presence of 1% sodium dodecyl sulfate, followed by preparative electrophoresis and reverse-polarity elution. However, these procedures resulted in loss of a large proportion of the activity, which precluded recovery of the protein in significant quality.
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; C051471:manganese-oxidizing protein, Leptothrix discophora; D008345:Manganese