The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria.
The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.
D000818:Animals; D002784:Cholesterol / Q000494:pharmacology; D003579:Cytochrome Reductases / Q000378:metabolism*; D003574:Cytochrome c Group; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D010740:Phospholipases / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology / Q000502:physiology*; D011485:Protein Binding; D051381:Rats; D012402:Rotenone / Q000494:pharmacology
D003574:Cytochrome c Group; D010743:Phospholipids; D012402:Rotenone; D002784:Cholesterol; D003579:Cytochrome Reductases; D010740:Phospholipases