Abstract
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.
Highlights
This study determines the validity of utilizing ra- [3].,despitea large body ofliterature relatedto these dioimmunoassayof CuZn and Mn superoxide dismutase enzymes in different organisms, bacteria [4], in the rat for defining mechanisms of control over there is onlylimited data available regardingthe mechanisms mammalian tissue superoxide dismutase concentra- of control over these enzymes in mammalian systems
The CuZn superoxide dismutase dimer had a specific activity of 3600 units/mg of protein and a subunit M, of 17,000.The Mn superoxide dismutase tetramer had a specific activity of 3700 units/mg of protein and a subunit M, of 22,000
Enzyme activity assays have proved less than ideal for this purpose, and theiruse has resulted inwidely varyingestimates of tissue Mn superoxidedismutase in the rat,a variation attributed to enzyme instability [5], low tissue content [6, 7],and to problems inherent to the enzyme activity assay systems [8]
Summary
This study determines the validity of utilizing ra- [3].,despitea large body ofliterature relatedto these dioimmunoassayof CuZn and Mn superoxide dismutase enzymes in different organisms, bacteria [4], in the rat for defining mechanisms of control over there is onlylimited data available regardingthe mechanisms mammalian tissue superoxide dismutase concentra- of control over these enzymes in mammalian systems. The CuZn superoxide dismutase dimer had a specific activity of 3600 units/mg of protein and a subunit M, of 17,000.The Mn superoxide dismutase tetramer had a specific activity of 3700 units/mg of protein and a subunit M, of 22,000 Both enzymes provided a single discrete proteinband on disc gel electrophoresis. To date no radioimmunoassay has tissue homogenate dilution and column chromatogra- been describedfor rat Mn superoxide dismutase, and phy Immunoassay of these enzymes in rat tissues permit- step, i.e. the purification of a stable rat Mn superoxide disted clarification of existing data based on activity as- mutase. Says and demonstrated a trend for higher Mn super- This manuscript details the methods for purification, laoxide dismutase concentrations in tissues of high mi- beling, and immunoassay of these rat enzymes and provides tochondrial content(withrelative tissue concentra- data on their stability and their concentrations invarious tions comparable to man) and low superoxide dismu- tissues.
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