Dataset Information

Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG.

ABSTRACT: Two forms of iron superoxide dismutase (SOD) were purified from cell extract (CE) and culture filtrate (CF) of Mycobacterium bovis BCG, respectively. The molecular weight of both enzymes was estimated to be approximately 84,000 by gel filtration, whereas that of their subunits was 21,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that each of purified enzymes is composed of four identical subunits. The specific activities of CE SOD and CF SOD were 3,850 and 4,040, respectively. The purified enzymes were not joined by disulfide bonds and were, to some extent, resistant to sodium dodecyl sulfate. Their activities were lost by H2O2, but not by azide and cyanide, indicating iron SODs. Enzyme activities were detectable over a broad range of pHs, from 5.0 to 9.0, and were stable for 6 months at -20 degreesC. Each value of pI was 4.5. In Western blots, both enzymes reacted with sera of tuberculosis patients, but not with normal sera. The N-terminal amino acid sequences of CE SOD and CF SOD were the same, suggesting that there is no N-terminal signal sequence.

SUBMITTER: Kang SK

PROVIDER: S-EPMC96202 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG.

Kang S K SK Jung Y J YJ Kim C H CH Song C Y CY

Clinical and diagnostic laboratory immunology 19981101 6

Two forms of iron superoxide dismutase (SOD) were purified from cell extract (CE) and culture filtrate (CF) of Mycobacterium bovis BCG, respectively. The molecular weight of both enzymes was estimated to be approximately 84,000 by gel filtration, whereas that of their subunits was 21,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that each of purified enzymes is composed of four identical subunits. The specific activities of CE SOD and CF SOD were 3,8 ...[more]

PMID: 9801335

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Project description:The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Dataset Information

Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG.

Publications

Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG.

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