Frånberg Jenny, Welander Hedvig, Aoki Mikio, Winblad Bengt, Tjernberg Lars O, Frykman Susanne
Karolinska Institutet Alzheimer's Disease Research Center (KI-ADRC), Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Novum plan 5, SE-141 57 Huddinge, Sweden.
Biochemistry. 2007 Jun 26;46(25):7647-54. doi: 10.1021/bi0621258. Epub 2007 May 31.
Gamma-secretase is important for the development of Alzheimer's disease, since it is a crucial enzyme for the generation of the pathogenic amyloid beta-peptide (Abeta). Most data on gamma-secretase is derived from studies in cell lines overexpressing gamma-secretase components or amyloid precursor protein (APP), and since gamma-secretase is a transmembrane protein complex, detergents have been frequently used to facilitate the studies. However, no extensive comparison of the influence of different detergents at different concentrations on gamma-secretase activity in preparations from brain has been made. Here, we establish the optimal conditions for gamma-secretase activity in rat brain, using an activity assay detecting endogenous production of the APP intracellular domain, which is generated when gamma-secretase cleaves the APP C-terminal fragments. We performed a subcellular fractionation and noted the highest gamma-secretase activity in the 100000g pellet and that the optimal pH was around 7. We found that gamma-secretase was active for at least 16 h at 37 degrees C and that the endogenous substrate levels were sufficient for activity measurements. The highest activity was obtained in 0.4% CHAPSO, which is slightly below the critical micelle concentration (0.5%) for this detergent, but the complex was not solubilized efficiently at this concentration. On the other hand, 1% CHAPSO solubilized a substantial amount of the gamma-secretase components, but the activity was low. The activity was fully restored by diluting the sample to 0.4% CHAPSO. Therefore, using 1% CHAPSO for solubilization and subsequently diluting the sample to 0.4% is an appropriate procedure for obtaining a soluble, highly active gamma-secretase from rat brain.
γ-分泌酶对阿尔茨海默病的发展至关重要，因为它是产生致病性淀粉样β肽（Aβ）的关键酶。关于γ-分泌酶的大多数数据来自对过表达γ-分泌酶成分或淀粉样前体蛋白（APP）的细胞系的研究，并且由于γ-分泌酶是一种跨膜蛋白复合物，去污剂经常被用于促进研究。然而，尚未对不同浓度的不同去污剂对脑提取物中γ-分泌酶活性的影响进行广泛比较。在这里，我们利用检测APP细胞内结构域内源性产生的活性测定法，建立了大鼠脑中γ-分泌酶活性的最佳条件，APP细胞内结构域是γ-分泌酶切割APP C末端片段时产生的。我们进行了亚细胞分级分离，发现100000g沉淀中的γ-分泌酶活性最高，最佳pH约为7。我们发现γ-分泌酶在37℃下至少16小时具有活性，并且内源性底物水平足以进行活性测量。在0.4% CHAPSO中获得了最高活性，这略低于该去污剂的临界胶束浓度（0.5%），但该复合物在此浓度下未有效溶解。另一方面，1% CHAPSO溶解了大量的γ-分泌酶成分，但活性较低。将样品稀释至0.4% CHAPSO可使活性完全恢复。因此，使用1% CHAPSO进行溶解，随后将样品稀释至0.4%，是从大鼠脑中获得可溶性、高活性γ-分泌酶的合适方法。
