PLANT PHYSIOLOGY , Vol 107, Issue 4 1139-1146, Copyright © 1995 by American Society of Plant Biologists
|
BIOCHEMISTRY AND ENZYMOLOGY |
2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases
FNJ. Droog, PJJ. Hooykaas and B. J. van der Zaal
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes
from tobacco (Nicotiana tabacum, White Burley) were partially
characterized. GST1-1 and GST2-1 are members of a recently identified new
type of plant GST isozymes that we will here refer to as type III. Both
enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when
expressed in bacteria as fusion proteins. The apparent Km for
1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM
for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for
glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The
in vitro activity of both enzymes could be inhibited by the synthetic auxin
2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus]
40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The
GST1-1 was also inhibited by structurally related substances, such as
2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated
structures benzoic acid and phenoxyacetic acid did not inhibit.
p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor,
was found to be an active inhibitor as well. The strongest inhibitor
identified, however, was a phenylacetic acid derivative, ethacrynic acid,
which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes.
This substance is a known inducer as well as a substrate of specific
mammalian GSTs. The results presented here indicate that the type III plant
GSTs might be involved in the metabolism or transport of chlorinated
substances that are structurally related to auxins. The possibility that
auxins are endogenous ligands or substrates for GSTs is discussed.
