Yeh L A, Cosgrove P, Holt W F
Department of Metabolic Diseases, Pfizer Central Research, Groton, CT 06385.
Membr Biochem. 1990 Apr-Jun;9(2):129-40. doi: 10.3109/09687689009025835.
A highly purified membrane fraction of H,K-ATPase was isolated from hog gastric mucosa by using differential centrifugation, sodium dodecyl sulfate (SDS:0.125%) treatment and density-gradient centrifugation. The final fraction showed a major band at 97 kD by SDS-gel electrophoresis. This purified H,K-ATPase sedimented at the interface of a 28-35% sucrose step gradient and displayed a specific activity of 140-170 mumol Pi/h/mg protein and a ratio of K-stimulated ATPase activity to Mg-stimulated ATPase activity of 6.5-8.7. The apparent Km for ATP was 0.154 mM and the Km for K+ was o.6 mM. The enzymatic activity recovered from this purification procedure was K(+)-ionophore-independent. SDS treatment in the presence of 2.5 mM ATP did not change the kinetic properties of the isolated enzyme. Exclusion of ATP during SDS solubilization diminished the enzymatic activity by 90%, indicating that ATP protection is essential for the full recovery of enzymatic activity. In summary, mild SDS solubilization can be used to purify relatively large quantities of active H,K-ATPase to near homogeneity without altering the enzyme's kinetic properties.
通过差速离心、十二烷基硫酸钠（SDS：0.125%）处理和密度梯度离心，从猪胃黏膜中分离出高度纯化的H，K - ATP酶膜组分。通过SDS - 凝胶电泳，最终组分在97 kD处显示出一条主要条带。这种纯化的H，K - ATP酶在28 - 35%蔗糖阶梯梯度的界面处沉淀，比活性为140 - 170 μmol Pi/h/mg蛋白质，K刺激的ATP酶活性与Mg刺激的ATP酶活性之比为6.5 - 8.7。ATP的表观Km为0.154 mM，K⁺的Km为0.6 mM。从该纯化过程中回收的酶活性不依赖于K⁺离子载体。在2.5 mM ATP存在下进行SDS处理不会改变分离酶的动力学性质。在SDS溶解过程中排除ATP会使酶活性降低90%，表明ATP保护对于酶活性的完全恢复至关重要。总之，温和的SDS溶解可用于纯化相对大量的活性H，K - ATP酶至接近均一性，而不会改变酶的动力学性质。
