Rat brain gamma-secretase activity is highly influenced by detergents.
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.
Frånberg
Jenny
J
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.
Welander
Hedvig
H
Aoki
Mikio
M
Winblad
Bengt
B
Tjernberg
Lars O
LO
Frykman
Susanne
S
D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D008297:Male; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature; D013997:Time Factors
D016564:Amyloid beta-Protein Precursor; D002793:Cholic Acids; D003902:Detergents; C048531:chapso; D053829:Amyloid Precursor Protein Secretases