Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms.
Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms.
复制标题
N,N-二环己基碳二亚胺对 NADH-泛醌还原酶活性的抑制以及这种抑制与各种生物体中能量偶联位点 1 的出现的相关性。
DOI：
10.1021/bi00384a025
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Yagi,T
中科院分区：
文献类型：
--
作者：
Yagi,T
材料和方法膜和分离的酶复合物的制备。大鼠肝线粒体通过使用Na-garse（Hatefi等人，1961年）。线粒体（36 mg/mL），在含有0.22 M甘露醇、0.07 M蔗糖、2 mM Ar-（2-羟乙基）哌嗪-A-2-乙磺酸（pH 7.4）和1%牛血清白蛋白，在最大功率下超声处理3次，持续1 min（50%脉冲），并在Spinco Model L 8 - 70 M的50 Ti转子中以16000 rpm离心5分钟。在50 Ti转子中以45 000 rpm再次离心沉淀物30 min。将沉淀悬浮于含有0.25 M蔗糖的10 mM Tris醋酸盐（pH 7.5）中，均质化，并储存在-80 ℃下。野生型S.酿酒酵母D273- 10 B在每升含有3g酵母提取物（Difco）、20 g半乳糖、lg KH 2 P 〇 4、0.5g CaCl 2·2 H20、lg NH 4Cl和0.6g MgCl 2·6 H20的培养基中生长。用50%（w/v）NaOH将最终pH调节至5.5。细胞需氧生长至对数中期。根据Daum等分离酵母线粒体，（1982年）。如针对大鼠肝SMP所述制备酵母亚线粒体颗粒。B。枯草杆菌细胞在含有17.5g/L细菌-抗生素培养基3（Difco，Detroit，MI）的培养基中需氧生长。在指数生长期结束时，收获细胞，并通过Konings等人的方法制备膜，（1973年）。将膜超声处理、离心、均质化并如上所述储存。如前所述制备P. acetificans膜和分离的副球菌NADH脱氢酶复合物（Yagi，1986）。
Materials and MethodsPreparation of Membranes and Isolated Enzyme Com-plexes. Rat liver mitochondria were prepared by using Na-garse (Hatefi et al., 1961). The mitochondria (36 mg/mL), in a solution containing 0.22 M mannitol, 0.07 M sucrose, 2 mM Ar-(2-hydroxyethyl) piperazine-A-2-ethanesulfonic acid (pH 7.4), and 1% bovine serum albumin, were sonicated 3 times at maximum power for 1 min (50% pulse) by a Branson sonifier and centrifuged for 5 min at 16000rpm in a 50 Ti rotor of Spinco Model L8-70M. Thesupernatant was re-centrifuged for 30 min at 45 000 rpm in the 50 Ti rotor. The pellet was suspended in 10 mM Tris-acetate (pH 7.5) con-taining 0.25 M sucrose, homogenized, and stored at-80 C. The wild-type S. cerevisiae D273-10B was grown in a medium containing per liter 3 g of yeast extract (Difco), 20 g of galactose, 1 g of KH2P04, 0.5 g of CaCl2-2H20, 1 g of NH4C1, and 0.6 g of MgCl2-6H20. The final pH was adjusted to 5.5 with 50%(w/v) NaOH. Cells were grown aerobically to mid-logarithmic phase. Yeast mitochondria were isolated according to Daum et al.,(1982). Yeast submitochondrial particles were prepared as described for rat liver SMP. B. subtilis cells were aerobically grown in a medium con-taining 17.5 g/L Bacto-antibiotic medium 3 (Difco, Detroit, MI). At the end of the exponential growth phase, the cells were harvested, and membranes were prepared by the methods of Konings et al.,(1973). The membranes were sonicated, centrifuged, homogenized, and stored as described above. P. denitrificans membranes and the isolated Paracoccus NADH dehydrogenase complex were prepared as described previously (Yagi, 1986).