Plant Physiol. (1978) 62, 468-469
Short Communication
2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane
NADH Dehydrogenase of Plant Mitochondria1
Received for publication April 20, 1978 and in revised form May 25, 1978
CARMEN A. MANNELLA
Edward A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, St. Louis, Missouri
63104
WALTER D. BONNER, JR.
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19174
ABSTRACT
The NADH dehydrogenase of potato (Solanum tuberosum) and mung
bean (Phaseolus aureus) outer mitochondrial membranes is specifically
inhibited by both 2,4-dchlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid.
The chemical agent 2,4-D is widely used as a selective herbicide
although its biochemical mode of action is not fully understood.
We have found that 2,4-D inhibits the NADH dehydrogenase
system associated with the outer membranes of potato (Solanum
tuberosum) and mung bean (Phaseolus aureus) mitochondria.
Figures I and 2 illustrate the inhibition by 2,4-D of the anti-
0.8-
006
0.
[NADHI] (mM)
FIG. 2. NADH dependence of outer mitochondrial membrane NADHCyt c oxidoreductase activity at different 2,4-D levels. Experimental
procedures as destribed in the legend to Figure 1. A; is the activity for
[NADHI 2 0.2 mm. (Note that A/AO = 0 when [NADHI = 0 for all 2,4-D
levels, so that the curves converge toward the [2,4-D] = 0 curve at NADH
concentrations below those shown here.)
o
mycin A-insensitive NADH-Cyt c oxidoreductase activity of outer
membranes isolated from potato mitochondria. When initial levels
oxidized Cyt c are 0.05 mm or higher, NADH concentrations
of
a 0.4\
below 0.2 mm are rate-controlling for this reaction sequence.
Inhibition by 2,4-D is detectable only at initial NADH levels
below this value (Fig. 2), indicating that 2,4-D acts at the dehydrogenase. The mode of inhibition of the NADH dehydrogenase
by 2,4-D is complex, appearing competitive with NADH at concentrations of this substrate just below 0.2 mm and uncompetitive
1.
0.4 0.8
at very low NADH concentrations (Fig. 2 and ref. 3). At an initial
[2,4-D] (mM)
NADH level of 0.1 mm, concentrations of 2,4-D approaching its
FIG. I. Inhibition of antimycin A-insensitive NADH-Cyt c oxidore- water solubility limit (about 2 mM) produce essentially complete
ductase activities by 2,4-D. Activities (A) measured as initial rate of Cyt inhibition of this outer mitochondrial membrane activity, while
c reduction (monitored with Zeiss PMQ ll spectrophotometer) upon inhibiting the microsomal NADH-Cyt c oxidoreductase to a far
addition of 0.1 mM NADH to membrane suspension consisting of approx- lesser extent (Fig. 1). It was found that the related compound
imately 50 ,ug of membrane protein, 0.05 mm oxidized Cyt c, I mM KCN, 2,4,5-trichlorophenoxyacetic acid is also a specific inhibitor of the
and 2.5 ,ug of antimycin A in 2.5 ml of 10 mM K-phosphate (pH 7.2). A/Ao outer mitochondrial membrane NADH dehydrogenase while
is the fractional activity of membranes incubated for 5 min in the indicated
concentrations of 2,4-D (Eastman) relative to the activity (Ao) in the IAA, the natural auxin whose hormonal action appears to be
absence of 2,4-D. Data presented for two different potato outer mitochon- mimicked by these chlorophenoxy acids, is not.
Since the function of the NADH dehydrogenase-Cyt bs,% system
drial membrane preparations (0, *) and for a preparation of potato light
microsomal membranes (El). Membrane fractions isolated according to of the outer mitochondrial membrane is not known, the physiological relevance of its inhibition by these compounds is unclear.
previously described procedures (1, 3).
A possibility suggested by these results is that this electron transfer
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involved, in plants, in auxin catabolism, inhibition of
systembyiswww.plantphysiol.org
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and the
National©Science
'Supported by grants from theCopyright
1978 American
Society
of Plant Biologists. All rights reserved.
which by 2,4-D could lead to elevated plant auxin levels and so
Herman Frasch Foundation.
468
Plant Physiol. Vol. 62, 1978
2,4-D INHIBITION OF NADH DEHYDROGENASE
might account for the herbicide's action. However, preliminary
experiments designed to detect either NADH oxidation or 02
utilization upon addition of IAA to concentrated suspensions of
potato or mung bean outer mitochondrial membranes have proven
negative (3).
The marked similarities between the NADH dehydrogenaseCyt b systems ofplant and animal outer mitochondrial membranes
(3, 5) raise the possibility that 2,4-D and 2,4,5-trichlorophenoxyacetic acid might inhibit the mammalian NADH dehydrogenase
as well. This should be checked, although present lack of understanding of the role of this enzyme system makes toxicological
assessment impossible. Along these lines, it has been shown that
these chlorophenoxy acids do not accumulate in human tissue
469
after single dose ingestion, although short term plasma levels in
human subjects of over 0.1 mm have been reported (2, 4).
LITERATURE CITED
I. DOUCE R, CA MANNELLA, WD BONNER JR 1973 The external NADH dehydrogenases of plant
mitochondria. Biochim Biophys Acta 292: 105- 116
2. GEHRING PJ, CG KRAMER, BA SCHWETZ, JG ROSE, VK ROWE 1973 The fate of 2,4,5trichlorophenoxyacetic acid (2,4,5-T) foUlowing oral administration to man. Toxicol Appl
Pharmacol 26: 352-361
3. MANNELLA CA 1974 Composition and structure of the outer membranes of plant mitochondria.
PhD thesis. Univ Pennsylvania, Philadelphia
4. SAUERHOFF MW, WH BRAUN, GC BLAU, PJ GEHRING 1977 The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. Toxicology 8: 3-1 1
5. SOTTOC.ASA GL, B KUYLENSTIERNA, L ERNSTER, A BERGSTRAND 1967 An electron transport
system associated with the outer membrane of liver mitochondria. J Cell Biol 32: 415-438
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.