Abstract
β-Secretase (β-SEC) is one of the prime targets for therapeutic intervention of Alzheimer’s disease. We previously described the expression of human β-SEC in Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST). To extend this work we investigated the kinetics of recombinant β-secretase and the feasibility of recombinant β-secretase for its inhibitor screening application. Recombinant β-SECs were purified from cultures of Tn5B1-4βSEC and Tn5B1-4βSEC/GalT-ST cells. Recombinant β-SEC from Tn5B1-4βSEC/GalT-ST cells exhibited a higher kcat/KM value compared to the control Tn5B1-4βSEC cells. We also attempted to screen the compounds inhibiting the activity of recombinant β-secretase among the metabolites isolated from Chrysanthemum coronarium L. Based on the inhibition analysis using purified β-SEC from Tn5B1-4βSEC/GalT-ST cells, 3-O-[β-D-galactopyranosyl (1→6)-α-D-galactopyranosyl]-glyceride was the best inhibitor among the compounds tested. The concentration at half-maximum inhibition values (IC50) of this compound was estimated to be 2.8 μM.
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