Immunohistochemical demonstration of an adrenal ferredoxin-like iron-sulfur protein in rat hepatic mitochondria.

Through the use of antibodies produced against homogeneous bovine adrenal ferredoxin, the presence of hepatic ferredoxin in rat hepatic mitochondria, its immunochemical similarity to adrenal ferredoxin, and its involvement in the hepatic mitochondrial cholesterol hydroxylase and NADPH-cytochrome c reductase activities have been demonstrated. Employing rabbit antiserum to bovine adrenal ferredoxin in an unlabeled antibody peroxidase-antiperoxidase technique, the presence of hepatic ferredoxin was detected immunohistochemically at the light microscopic level in all parenchymal cells within the liver lobule as well as in preparations of isolated rat hepatic mitochondria. Immunohistochemical staining for hepatic ferredoxin was not detected in rat hepatic microsomal preparations. The presence of hepatic ferredoxin in rat hepatic mitochondria and its immunochemical similarity to adrenal ferredoxin were further verified by EPR spectroscopy. Upon the addition of sodium dithionite to a concentrated supernatant fraction prepared from sonicated rat hepatic mitochondria, an EPR spectrum characteristic of adrenal ferredoxin-like iron-sulfur proteins was observed at 100 K (gX = g, = 1.935 and g, = 2.02). The magnitude of theg = 1.935 signal of reduced hepatic ferredoxin was observed to be diminished in the presence of goat antibody to bovine adrenal ferredoxin. Moreover, this antibody was also found to be capable of inhibiting both cholesterol hydroxylation and the NADPH-dependent reduction of cytochrome c catalyzed by the rat hepatic mitochondrial supernatant fraction, thereby demonstrating the involvement of hepatic ferredoxin in these two enzymatic activities.