Barber M J, Zektzer A S, Rosen G M, Dëmos H A, Rauckman E J
Biochim Biophys Acta. 1984 Sep 19;776(1):159-68. doi: 10.1016/0005-2736(84)90262-1.
Hepatic microsomal membranes, prepared under various conditions that yield either 'intact' or 'disrupted' microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of N-ethylmaleimide. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the 'W/S' ratio. For latent microsomes, the value of this parameter was determined to be 0.65 +/- 0.02 and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The W/S ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The W/S ratio was observed to be dependent upon the method of microsome preparation yielding values of 1.02 +/- 0.02 for 'hypertonically disrupted' vesicles and 1.28 +/- 0.02 for 'mechanically disrupted' vesicles. Microsomal marker enzymes such as cytochrome P-450 and FAD-containing monooxygenase retained significant levels of functionality following nitroxide incorporation.
在各种条件下制备的肝微粒体膜，可产生“完整”或“破裂”的微粒体囊泡，已使用N - 乙基马来酰亚胺的氮氧化物类似物通过内在膜蛋白的巯基进行标记。电子顺磁共振光谱显示存在两类主要的结合标记，对应于不同程度的固定化，使用称为“W/S”比的参数对其比例进行了定量。对于潜伏微粒体，该参数的值确定为0.65±0.02，并受标记/蛋白质比、孵育时间、氮氧化物结构、温度和pH等因素影响。如葡萄糖 - 6 - 磷酸水解酶的甘露糖6 - 磷酸活性的潜伏性所示，W/S比也对膜完整性程度敏感。此外，膜破裂导致插入脂质双层中的氮氧化物标记脂肪酸的序参数相应降低。观察到W/S比取决于微粒体制备方法，“高渗破裂”囊泡的值为1.02±0.02，“机械破裂”囊泡的值为1.28±0.02。在掺入氮氧化物后，微粒体标记酶如细胞色素P - 450和含FAD的单加氧酶保留了显著水平的功能。
