Cloning and characterization of a glutathione S-transferase that can be photolabeled with 5-azido-indole-3-acetic acid.
Previously, we identified a soluble protein from Hyoscyamus muticus that was photolabeled by 5-azido-indole-3-acetic acid. This protein was determined to be a glutathione S-transferase (GST; J. Bilang, H. Macdonald, P.J. King, and A. Sturm [1993] Plant Physiol 102: 29-34). We have examined the effect of auxin on the activity of this H. muticus GST. Auxins reduced enzyme activity only at high concentrations, with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid being more effective than indole-3-acetic acid (IAA) and naphthylacetic acid. IAA was a noncompetitive inhibitor, whereas inhibition by 2,4-D was competitive with respect to 1-chloro-2,4-dinitro-benzene. We also present the sequence of a full-length cDNA clone that codes for a GST and contains all partial amino acid sequences of the purified protein. The auxin-binding GST was found in high amounts in roots and stems and low amounts in leaves and flower buds. The steady-state mRNA level was not regulated by IAA or naphthylacetic acid, whereas 2,4-D and 2,3-dichlorophenoxyacetic acid increased mRNA levels. We propose a model in which 2,4-D is a substrate for GST, whereas IAA binds at a second site, known as a ligandin-binding site for the purpose of intracellular transport.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D010777:Photochemistry; D010944:Plants / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D001386:Azides; D018076:DNA, Complementary; D018744:DNA, Plant; D006540:Herbicides; D007210:Indoleacetic Acids; D012333:RNA, Messenger; D018749:RNA, Plant; C068520:5-azido-1H-indole-3-acetic acid; D005982:Glutathione Transferase