An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

Abstract

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADHcytochrome b5 reductase and cytochrome bs.

Human hepatic and renal microsomes, cytochromes P450 1A1/2, NADPH:Cytochrome P450 reductase and prostaglandin H synthase mediate the formation of aristolochic acid‐DNA adducts found in patients with urothelial cancer

“…The renal microsomal sample was analyzed only for specific content of CYP and specific activity of CYP1A (ethoxyresorufin O-deethylation). The activity of NADPH:CYP reductase in human hepatic and renal microsomes was measured according to Sottocasa et al 41 using cytochrome c as substrate (i.e., as NADPH: cytochrome c reductase). The concentration of NADPH:CYP reductase was estimated as described previously.…”

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

“…Pi liberated in the above essays was determined as described by Martin & Doty (1949). NADPH-cytochrome c oxidoreductase activity (EC 1.6.2.4) was assayed by the method of Sottocasa et al (1967). Succinate dehydrogenase activity (EC 1.3.99.1) was assayed by the procedure of Earl & Korner (1965).…”

Vitamin C restores healthy aging in a mouse model for Werner syndrome

“…The cytochrome-b5-reductase activity was measured as described previously (27,28). Briefly, liver tissues were homogenized in ice-cold 0.25 M sucrose and 10 mM HEPESNaOH, pH 7.8 (iso-osmotic buffer, 4 ml/g of liver) with a cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA).…”

“…The protein concentration of the sample was determined by the standard Bradford assay. The enzymatic reaction was carried out in a 50 mM phosphate buffer, pH 7.5 (27,28), in the presence of 0.3 mM KCN (to inhibit oxidases) and 2 μM rotenone (to inhibit mitochondrial NADH cytochrome C reductases). The reaction was started by the addition of 0.5 mM NADH.…”