Presence of the NADPH-Cytochrome P-450 Reductase System in Liver and Kidney Mitochondria

Abstract

Sonic extracts of bovine liver and kidney mitochondria showed a weak but definite NADPH-chytochrome c reductase activity. 80% of the total reductase activity was due to contamination by microsomal NADPH-cytochrome c reductase, as shown by inhibition of the microsomal reductase with the antibody. However, the residual NADPH-cytochrome c reductase activity of mitochondrial extracts was intrinsic to mitochondria. The NADPH-cytochrome c reductase activity of these mitochondrial sonic extracts was stimulated by the addition of adrenodoxin reductase, which had been purified from adrenal cortex mitochondria, and the increase of the reductase activity was proportional to the amount of adrenodoxin reductase added. The antibody against pure adrenodoxin inhibited aderenodoxin reductase-dependent NADPH-cytochrome c reductase activity almost completely. The presence of adrenodoxin in liver and kidney mitochondrial extracts was further confirmed by Ouchterlony double diffusion and quantitative immunoprecipitation. When the sonic extracts of liver and kidney mitochondria were titrated with adrenodoxin which had been purified from bovine adrenal cortex mitochondria, NADPH-cytochrome c reductase activity was enhanced in proportion to the amount of adrenodoxin added. Although the antibody against pure adrenodoxin reductase did not inhibit the catalytic activity of the reductase, specific removal of the reductase in mitochondrial extracts by immunoprecipitation was utilized to demonstrate the immunochemical identity of the component in liver and kidney mitochondria with adrenodoxin reductase. Their identity was confirmed by Ouchterlony double diffusion. Thus, the NADPH-cytochrome c reductase activity in liver and kidney mitochondrial extracts is composed of adrenodoxin and adrenodoxin reductase, which are both molecularly identical with those in adrenal cortex mitochondria; these two mitochondrial components may function in the hydroxylation of steroid compounds in liver and kidney mitochondria as an NADPH-cytochrome P-450-reducing system.