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.
Full text
PDF






Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abramovitz A. S., Randolph V., Mehra A., Christakos S. Recovery of native proteins from preparative electrophoresis gel slices by reverse polarity elution. Prep Biochem. 1984 Aug;14(3):205–221. doi: 10.1080/10826068408070629. [DOI] [PubMed] [Google Scholar]
- Adams L. F., Ghiorse W. C. Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora. Appl Environ Microbiol. 1985 Mar;49(3):556–562. doi: 10.1128/aem.49.3.556-562.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ali S. H., Stokes J. L. Stimulation of heterotrophic and autotrophic growth of Sphaerotilus discophorus by manganous ions. Antonie Van Leeuwenhoek. 1971;37(4):519–528. doi: 10.1007/BF02218522. [DOI] [PubMed] [Google Scholar]
- Boogerd F. C., de Vrind J. P. Manganese oxidation by Leptothrix discophora. J Bacteriol. 1987 Feb;169(2):489–494. doi: 10.1128/jb.169.2.489-494.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Ghiorse W. C. Biology of iron- and manganese-depositing bacteria. Annu Rev Microbiol. 1984;38:515–550. doi: 10.1146/annurev.mi.38.100184.002503. [DOI] [PubMed] [Google Scholar]
- Granelli-Piperno A., Reich E. A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med. 1978 Jul 1;148(1):223–234. doi: 10.1084/jem.148.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hajj H., Makemson J. Determination of growth of Sphaerotilus discophorus in the presence of manganese. Appl Environ Microbiol. 1976 Nov;32(5):699–702. doi: 10.1128/aem.32.5.699-702.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jung W. K., Schweisfurth R. Manganese oxidation by an intracellular protein of a Pseudomonas species. Z Allg Mikrobiol. 1979;19(2):107–115. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Rosson R. A., Tebo B. M., Nealson K. H. Use of poisons in determination of microbial manganese binding rates in seawater. Appl Environ Microbiol. 1984 Apr;47(4):740–745. doi: 10.1128/aem.47.4.740-745.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]
- van Veen W. L., Mulder E. G., Deinema M. H. The Sphaerotilus-Leptothrix group of bacteria. Microbiol Rev. 1978 Jun;42(2):329–356. doi: 10.1128/mr.42.2.329-356.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]