Mamelok R D, Tse S S, Newcomb K, Bildstein C L, Liu D
Biochim Biophys Acta. 1982 Oct 22;692(1):115-25. doi: 10.1016/0005-2736(82)90509-0.
Basal-lateral membranes from the renal cortex of the rabbit were isolated by sucrose gradient centrifugation in a zonal rotor which allows for a large-scale preparation of these membranes. A heterogeneous population of membranes (P4) which contained 29% of the (Na+ + K+)-ATPase found in the homogenate of renal cortex was prepared by differential centrifugation. When pellet P4 was subjected to centrifugation in a sucrose gradient the activity of (Na+ + K+)-ATPase, a marker for basal-lateral membranes, could be separated from enzymatic markers of other organelles. The specific activity of (Na+ + K+)-ATPase was enriched 12-fold at a density of 1.141 g/cm3. Membranes (P alpha) contained in the (Na+ + K+)-ATPase-rich fractions consisted primarily of closed vesicles which exhibited probenecid inhibitable transport of rho-aminohippurate. These membranes did not exhibit Na+-dependent, phlorizin-inhibitable D-glucose transport. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from P alpha revealed at least six major protein bands with molecular weights of 91000, 81000, 73000, 65000, 47000 and 38000. A small fraction of total alkaline phosphatase found in the homogenate was found in pellet P4. Membranes containing this alkaline phosphatase activity were distributed widely over the gradient, with peak activity found at a density of 1.141 g/cm3. In contrast, when brush borders were subjected to gradient centrifugation under the same conditions as P4, alkaline phosphatase was found in a narrow distribution, with peak activity at a density of 1.158 g/cm3. The principle subcellular localization of the alkaline phosphatase found in P4 could not be determined unambiguously from the data, but the activity did not seem to be primarily associated with classical brush borders.
通过在区带转子中进行蔗糖梯度离心，从兔肾皮质分离出基底外侧膜，这种方法可大规模制备这些膜。通过差速离心制备了一种异质膜群体（P4），其含有肾皮质匀浆中29%的（Na⁺+K⁺）-ATP酶。当沉淀P4在蔗糖梯度中离心时，作为基底外侧膜标志物的（Na⁺+K⁺）-ATP酶活性可与其他细胞器的酶标志物分离。（Na⁺+K⁺）-ATP酶的比活性在密度为1.141 g/cm³时富集了12倍。富含（Na⁺+K⁺）-ATP酶的组分中所含的膜（Pα）主要由封闭小泡组成，这些小泡表现出丙磺舒可抑制的对氨基马尿酸转运。这些膜未表现出Na⁺依赖性、根皮苷抑制的D-葡萄糖转运。对来自Pα的蛋白质进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳，显示至少有六条主要蛋白带，分子量分别为91000、81000、73000、65000、47000和38000。在匀浆中发现的总碱性磷酸酶的一小部分存在于沉淀P4中。含有这种碱性磷酸酶活性的膜在梯度中分布广泛，在密度为1.141 g/cm³时活性达到峰值。相比之下，当刷状缘在与P4相同的条件下进行梯度离心时，碱性磷酸酶分布狭窄，在密度为1.158 g/cm³时活性达到峰值。从这些数据中无法明确确定P4中发现的碱性磷酸酶的主要亚细胞定位，但该活性似乎主要不与经典刷状缘相关。
