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 Ny codenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutas e (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former acti vity and by the use of specific antibodies. As expected, the cytosol and ly sosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix conta ined Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane spac e and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD assoc iated with the inner membrane was solubilized by 0.5 M NaCl. Surprisingly t he intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD t hat 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 com pared. They have identical molecular mass, cyanide sensitivity, SDS sensiti vity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn -SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cy tosolic and the intermembrane space Cu,Zn-SODS are coded for by the same ge ne. The significance of this distribution of the SODS is discussed.