Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1

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• 作者：Johan Å ; l ; er ; Katarina Johansson ; Vanina Dahlströ ; m Heuser ; Henny Farebo ; Julia Jä ; rvliden ; Hiroshi Abe ; Aya Shibata ; Mika Ito ; Yoshihiro Ito ; Ralf Morgenstern
• 关键词：Fluorescent substrate ; MGST1 ; Glutathione transferase
• 刊名：Analytical Biochemistry
• 出版年：2009
• 出版时间：1 July 2009
• 年：2009
• 卷：390
• 期：1
• 页码：52-56
• 全文大小：826 K

文摘

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246–2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k_cat = 0.075 ± 0.005 s⁻¹ and K_m = 21 ± 3 μM (k_cat/K_m = 3.6 × 10³ ± 5.6 × 10² M⁻¹ s⁻¹), giving a rate enhancement of 10⁶ compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.