Rat superoxide dismutases. Purification, labeling, immunoassay, and tissue concentration

This study determines the validity of utilizing radioimmunoassay of CuZn and Mn superoxide dismutase in the rat for defining mechanism of control over mammalian tissue superoxide dismutase concentrations. To accomplish this, rat Mn and CuZn superoxide dismutase were purified. The CuZn superoxide dis...

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Bibliographic Details
Published in:The Journal of biological chemistry 1985-02, Vol.260 (4), p.2212-2217
Main Authors: Asayama, K, Burr, I M
Format: Article
Language:English
Subjects:
Animals
Drug Stability
Kidney - enzymology
Liver - enzymology
Lung - enzymology
Male
Muscles - enzymology
Myocardium - enzymology
Pancreas - enzymology
Radioimmunoassay
Rats
Rats, Inbred Strains
Superoxide Dismutase - analysis
Tissue Distribution
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Summary:This study determines the validity of utilizing radioimmunoassay of CuZn and Mn superoxide dismutase in the rat for defining mechanism of control over mammalian tissue superoxide dismutase concentrations. To accomplish this, rat Mn and CuZn superoxide dismutase were purified. The CuZn superoxide dismutase dimer had a specific activity of 3600 units/mg of protein and a subunit Mr of 17,000. The Mn superoxide dismutase tetramer had a specific activity of 3700 units/mg of protein and a subunit Mr of 22,000. Both enzymes provided a single discrete protein band on disc gel electrophoresis. The purified enzymes were utilized to develop sensitive (less than 2.5 ng/ml Mn superoxide dismutase and less than 3.12 ng/ml CuZn superoxide dismutase) reproducible immunoassays the specificity of which was confirmed by tissue homogenate dilution and column chromatography. Immunoassay of these enzymes in rat tissues permitted clarification of existing data based on activity assays and demonstrated a trend for higher Mn superoxide dismutase concentrations in tissues of high mitochondrial content (with relative tissue concentrations comparable to man) and low superoxide dismutase concentrations in islets (providing an explanation for their sensitivity to free radical damage). This represents the first report of a radioimmunoassay for rat Mn superoxide dismutase, and the second report of successful purification of rat Mn superoxide dismutase (with higher specific activity and apparent purity and stability). The data support the proposition that these radioimmunoassays in rats will provide a useful system for investigation of mechanisms of control over tissue superoxide dismutase concentrations in mammalian tissues.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)89540-0