期刊
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
卷 34, 期 4, 页码 321-329出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.ibmb.2003.12.001
关键词
glutathione S-transferases; Bemisia tabaci; molecular cloning
Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and mono-chlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 muM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single hand on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs. (C) 2003 Elsevier Ltd. All rights reserved.
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