Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae).
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 monochlorobimane (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 microM 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 band 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.
Rauch
Natascha
N
Bayer CropScience AG, Research, Global Biology Insecticides, Alfred Nobel Str. 50, D-40789 Monheim, Germany.
Nauen
Ralf
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D001728:Dicumarol / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006430:Hemiptera / Q000201:enzymology* / Q000235:genetics; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011720:Pyrazoles / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D013045:Species Specificity; D013379:Substrate Specificity
D018076:DNA, Complementary; D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011994:Recombinant Proteins; C059597:monochlorobimane; D001728:Dicumarol; D005982:Glutathione Transferase; D004976:Ethacrynic Acid