Development of Evaluation System of β-secretase Activity by Using Orientated Immobilizing Recombinant β-secretase

Alzheimer's disease (AD) is one of the type of dementia and the numbers of the patients has been increasing in the world. However, there are no effective reagents for the disease because the cause of AD is not well-known. The neuropathological diagnosis of AD is decided upon the detection of amyloid plaques which made by insoluble amyloid. It is known that the amyloid is produced by β-secretase and γ-secretase which catalyze the amyloid precursor peptide. The β-Secretase is known as a one of a key enzyme for development of therapeutic agent to AD because the enzyme activity is specifically high in AD patients. All the conventional β-secretase inhibitors are substrate analogs, it means that those inhibitors are competitive and reversible inhibitor. Therefore, a non competitive enzyme inhibitor of β-secretase will be able to be a novel reagent as an anti-AD. In the previous study, we already developed the system to evaluate properties of the inhibitors whether its effect is reversible or irreversible. In this study, we developed evaluation system for activity of β-secretase by using immobilized recombinant β-secretase in combination with a FIA (Flow Injection Analysis) system. On the other hand, conventional method for enzyme immobilization cannot control enzyme orientation. Hence we investigated orientation of immobilized enzyme by using specific binding of biotin and streptavidin. And we tried to miniaturize the system to reduce assay time and increase the sensitivity. Furthermore, we applied the system to evaluate the property of conventional inhibitor.