Varga S, Szabolcs M
F. Verzár International Laboratory for Experimental Gerontology, Hungarian Section.
Acta Physiol Hung. 1994;82(2):139-52.
Multilamellar 3-dimensional (Type I) crystals of detergent-solubilized, purified (Na+, K+)-ATPase enzyme of pig kidney grow in media consisting of 0.1 M KCl, 0.1 M NaCl, 20 mM imidazole pH: 7.5 (20 degrees C), 5 mM MgCl2, 5 mM DTT, 3 mM ZNaN3, 0.025 TIU/ml Aprotinin, 2 micrograms/ml BHT and 20-40% glycerol, using nonionic detergents of C12E8 or BRIJ 36 for solubilization. The refined crystallization protocol: the use of media containing 20% glycerol, the low detergent: protein ratio and preincubation at subzero temperature at the initial phase of crystallization resulted in a remarkable increase of the yield and overall dimension of the crystals (up to 3-4 microns), while the stacking of the crystalline sheets was dramatically reduced. Biochemical and structural analysis of these crystals revealed further similarities between the 3-d crystals of the (Na+, K+)-ATPase and the Ca2+ ATPase of skeletal muscle-SR (Taylor and Varga, J. Biol. Chem., 269, 10107-10111, 1994). Computer image processing of the electron micrographs of stacked crystalline sheets of (Na+,K+)-ATPase molecules gave unit cell dimensions: a = 166.2 +/- 3.8 A, b = 54.2 +/- 3.5 A, with an included angle of 90 degrees C. Based on the close identity of the filtered images in projection and of other data, we concluded that the 3-dimensional crystals of the (Na+, K+)-ATPase contain only the alpha catalytic subunits.
猪肾中经去污剂增溶、纯化的（Na⁺，K⁺）-ATP 酶的多层三维（I 型）晶体，在含有 0.1M KCl、0.1M NaCl、20mM 咪唑（pH：7.5，20℃）、5mM MgCl₂、5mM 二硫苏糖醇（DTT）、3mM 叠氮化钠（NaN₃）、0.025 胰蛋白酶抑制单位/毫升抑肽酶、2 微克/毫升丁基羟基甲苯（BHT）和 20 - 40%甘油的培养基中生长，使用 C12E8 或 BRIJ 36 等非离子去污剂进行增溶。优化后的结晶方案：使用含 20%甘油的培养基、低去污剂与蛋白质比例以及在结晶初始阶段进行零下温度预孵育，使晶体产量和整体尺寸显著增加（可达 3 - 4 微米），同时晶体片层的堆叠显著减少。对这些晶体的生化和结构分析揭示了（Na⁺，K⁺）-ATP 酶的三维晶体与骨骼肌 - 肌浆网（SR）的 Ca²⁺ - ATP 酶之间的进一步相似性（泰勒和瓦尔加，《生物化学杂志》，269，10107 - 10111，1994）。对（Na⁺，K⁺）-ATP 酶分子堆叠晶体片层的电子显微照片进行计算机图像处理，得到晶胞尺寸：a = 166.2 ± 3.8 Å，b = 54.2 ± 3.5 Å，夹角为 90℃。基于投影中滤波图像的紧密一致性及其他数据，我们得出结论，（Na⁺，K⁺）-ATP 酶的三维晶体仅包含α催化亚基。
