Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria.
Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.
Okado-Matsumoto
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Fridovich
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I
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003486:Cyanides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008928:Mitochondria / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000737:chemistry*; D013997:Time Factors
D003486:Cyanides; D003902:Detergents; D012967:Sodium Dodecyl Sulfate; D013482:Superoxide Dismutase