Rivera-Morán Miguel A, Sampedro José G
Instituto de Física, Universidad Autónoma de San Luis Potosí, Avenida Chapultepec 1570, Privadas del Pedregal, San Luis Potosí 78295, Mexico.
Methods Protoc. 2023 Oct 19;6(5):102. doi: 10.3390/mps6050102.
The sarcoendoplasmic reticulum Ca-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca and the enzyme kinetics showed a hyperbolic dependence on ATP ( = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (). The presence of Ca did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.
肌浆网钙 - ATP酶（SERCA）是一种膜蛋白，在缺乏钙离子的纯化过程中会变得不稳定。二糖海藻糖是一种蛋白质稳定剂，在应激状态下会在酵母细胞质中积累。在本研究中，通过在纯化方案中加入海藻糖来纯化SERCA。纯化后的SERCA显示出高蛋白纯度（约95%）和ATP酶活性。ATP水解依赖于钙离子的存在，并且酶动力学显示对ATP呈双曲线依赖性（ = 12.16 ± 2.25 μM ATP）。FITC标记显示了ATP结合位点的完整性以及所分离的酶作为P型ATP酶的特性。用ATP滴定后，在225 nm波长处观察到圆二色性（CD）光谱变化，表明核苷酸结合结构域（N - 结构域）中的α - 螺旋重排，这与ATP亲和力相关（）。钙离子的存在不影响FITC标记或N - 结构域处ATP介导的结构变化。在SERCA纯化方案中使用海藻糖可使酶稳定。所分离的SERCA似乎适用于结构和配体结合研究，例如用于测试新设计的或天然的抑制剂。建议使用海藻糖来分离不稳定的酶。
