Varga S
F. Verzar International Laboratory for Experimental Gerontology, University Medical School, Debrecen, Hungary.
Acta Physiol Hung. 1994;82(4):365-76.
Multilamellar 3-dimensional (Type I) microcrystals of detergent-solubilized crude microsomes or purified protein preparations of membrane-bound gastric (H+, K+)-ATPase from rabbit or hog stomachs develop in media consisting of 0.1 M KCl, 20 mM imidazole, 5 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml DTBpC and 20-40% glycerol, using nonionic detergent of C12E8 or BRIJ 36 for solubilization. Crystals developed in a pH-range of 6.0-7.25, during 3-10 days of incubation, at 2 degrees C. For C12E8, the most effective detergent:protein ratio for crystallization varied between (1.8-2.0):1 for the microsomes and between (0.25-0.75):1 for the purified preparations. The results of biochemical and structural analysis of the (H+, K+)-ATPase crystals showed close resemblance to those of the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle and plasmamembrane (Na+, K+)-ATPase from kidney (J. Biol. Chem., 269, 10107-111, 1994). Based on these identities and the high (62%) overall sequential homology to the (Na+, K+)-ATPase, we conclude that the new crystals of the (H+, K+)-ATPase could also contain only the alpha-chain of the alpha beta heterodimers found in the native membrane. High-resolution electron microscopy of frozen-hydrated crystalline (H+, K+)-ATPase samples are in progress to give unit cell dimensions and molecular packing of the new crystals.
用C12E8或BRIJ 36非离子去污剂溶解来自兔或猪胃的去污剂溶解的粗微粒体或膜结合胃（H⁺，K⁺）-ATP酶的纯化蛋白制剂，在由0.1M KCl、20mM咪唑、5mM MgCl₂、3mM叠氮化钠、5mM二硫苏糖醇、25IU/ml抑肽酶、2μg/ml二丁基邻苯二甲酰亚胺和20-40%甘油组成的培养基中形成多层三维（I型）微晶。晶体在pH值6.0-7.25范围内，于2℃孵育3-10天期间形成。对于C12E8，微晶化最有效的去污剂与蛋白质比例，微粒体为（1.8-2.0）：1，纯化制剂为（0.25-0.75）：1。（H⁺，K⁺）-ATP酶晶体的生化和结构分析结果与骨骼肌肌浆网Ca²⁺-ATP酶和肾质膜（Na⁺，K⁺）-ATP酶的结果非常相似（《生物化学杂志》，269，10107-111，1994）。基于这些一致性以及与（Na⁺，K⁺）-ATP酶62%的总体序列同源性，我们得出结论，（H⁺，K⁺）-ATP酶的新晶体也可能仅包含天然膜中发现的αβ异二聚体的α链。对冷冻水合结晶（H⁺，K⁺）-ATP酶样品的高分辨率电子显微镜研究正在进行中，以给出新晶体的晶胞尺寸和分子堆积情况。
