Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1.
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 microM (k(cat)/K(m)=3.6 x 10(3)+/-5.6 x 10(2)M(-1)s(-1)), giving a rate enhancement of 10(6) 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.
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, SE-17177 Stockholm, Sweden.
Johan
J
Alander
Katarina
K
Johansson
Vanina Dahlström
VD
Heuser
Henny
H
Farebo
Julia
J
Järvliden
Hiroshi
H
Abe
Aya
A
Shibata
Mika
M
Ito
Yoshihiro
Y
Ito
Ralf
R
Morgenstern
D000818:Animals; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D012235:Rhodamines; D013449:Sulfonamides; C542491:bis-2,4-dinitrobenzenesulfonyl rhodamine; D005982:Glutathione Transferase