KMID : 0903520090520020109
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology 2009 Volume.52 No. 2 p.109 ~ p.113
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Kinetics of Recombinant ¥â-Secretase and Its Application to Inhibitor Screening
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Seo Hea-Kyung
Yoo Ki-Hyun Jeon Hwang-Bo Seok Yeon-Ju Kim Kyung-Il Baek Nam-In Lee Youn-Hyung Yang Jai-Myung Chung In-Sik
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Abstract
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¥â-Secretase (¥â-SEC) is one of the prime targets for therapeutic intervention of Alzheimer¡¯s disease. We previously described the expression of human ¥â-SEC in Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding ¥â1,4-galactosyltransferase (GalT) and Gal¥â1,4-GlcNAc ¥á2,6-sialyltransferase (ST). To extend this work we investigated the kinetics of recombinant ¥â-secretase and the feasibility of recombinant ¥â-secretase for its inhibitor screening application. Recombinant ¥â-SECs were purified from cultures of Tn5B1-4¥âSEC and Tn5B1-4¥âSEC/GalT-ST cells. Recombinant ¥â-SEC from Tn5B1-4¥âSEC/GalT-ST cells exhibited a higher kcat/KM value compared to the control Tn5B1-4¥âSEC cells. We also attempted to screen the compounds inhibiting the activity of recombinant ¥â-secretase among the metabolites isolated from Chrysanthemum coronarium L. Based on the inhibition analysis using purified ¥â-SEC from Tn5B1-4¥âSEC/GalT-ST cells, 3-O-[¥â-D-galactopyranosyl (1¡æ6)-¥á-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 ¥ìM.
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KEYWORD
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enzymatic activity, recombinant ¥â-secretase, Trichoplusia ni Tn5B1-4 cells, 3-O-[¥â-Dgalactopyranosyl
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