Feo F, Canuto R A, Garcea R, Brossa O
Biochim Biophys Acta. 1978 Oct 11;504(1):1-14. doi: 10.1016/0005-2728(78)90002-6.
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.
通过丙酮提取、磷脂酶A2或磷脂酶C消化诱导的大鼠肝线粒体磷脂耗竭，极大地抑制了NADH-细胞色素c还原酶（鱼藤酮不敏感型）的活性。用磷脂酶A2孵育后，分离的线粒体外膜中的还原酶活性也大幅下降。向脂质缺乏的线粒体或脂质缺乏的外膜中添加线粒体磷脂混合物，酶活性几乎完全恢复。线粒体外膜中存在的单个磷脂对还原酶活性几乎没有或没有刺激作用。鸡蛋磷脂酰胆碱是最具活性的磷脂，但二棕榈酰磷脂酰胆碱几乎无效。线粒体的脂质耗竭导致了表征天然还原酶活性的非线性阿伦尼乌斯曲线消失。用线粒体磷脂重构的酶呈现出与天然酶几乎相同的非线性曲线。在脂质缺乏的线粒体中，Triton X-100、吐温80或脱氧胆酸钠仅诱导NADH-细胞色素c还原酶（鱼藤酮不敏感型）产生少量激活。以1:1摩尔比向提取的线粒体磷脂中添加胆固醇会抑制NADH-细胞色素c还原酶（鱼藤酮不敏感型）的再激活，但不影响磷脂与脂质缺乏的线粒体或脂质缺乏的外膜的结合。这些结果表明，线粒体外膜的NADH-细胞色素c还原酶（鱼藤酮不敏感型）的活性需要磷脂。磷脂混合物比单个磷脂或去污剂更能满足这一需求。似乎膜流动性也可能影响还原酶活性。
