Abstract-DIMBOA (2,4-dihydroxy-7-methoxy-l,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits energy transfer reactions. In this paper the effect of DIMBOA on the ATPase activity of chloroplast coupling factor 1 (CF,) was studied. A rapid inactivation of the enzyme was observed which increased with the concentration of DIMBOA. This inactivation was reversed by dilution and by filtration of the inhibitor. The inactivation was partially prevented by pre-treatment of the enzyme with iodoacetamide. A slower simultaneous inactivation was observed which followed pseudo-first order kinetics. It is suggested that part of the rapid inhibition of CF, ATPase caused by DIMBOA is due to its reaction with sulthydryl groups on the enzyme. These reactions of DIMBOA may explain some of the inhibitory properties of DIMBOA on a wide range of organisms

Carlos B Queirolo

Carlos S Andreo

Hermann M Niemeyer*$

Luis J Corcuera

CiteSeerX

Phytochemistry, Vol. 22, No. 11, pp. 2455-2458, 1983 
Printed in Great Britain. 
0031-9422183 %3.00+0.00 
0 1983 Pergamon Press Ltd. 
INHIBITION OF ATPase FROM CHLOROPLASTS BY A HYDROXAMIC ACID 
FROM THE GRAMINEAE 
CARLOS B. QUEIROLO,* CARLOS S. ANDREo,t HERMANN M. NIEMEYER*$ and LUIS J. CORCUERA* 
l Facultad de Ciencias Bgsicas y Farmacbticas, Universidad de Chile, Casilla 653, Santiago, Chile; tCentro de Estudios 
Fotosint&ticos y Bioquimicos (Consejo National de Investigaciones Cientificas y Tkcnicas, Fundaci6n Miguel Lillo and Universidad 
National de Rosario), Suipacha 531, 2000 Rosario, Argentina 
(Revised received 21 February 1983) 
Key Word Indexaramineae; resistance factor; 1,4-benzoxazin-3-ones; chloroplast coupling factor; thiols. 
Abstract-DIMBOA (2,4-dihydroxy-7-methoxy-l,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae 
involved in the resistance of cereals to aphids, inhibits energy transfer reactions. In this paper the effect of DIMBOA on 
the ATPase activity of chloroplast coupling factor 1 (CF,) was studied. A rapid inactivation of the enzyme was 
observed which increased with the concentration of DIMBOA. This inactivation was reversed by dilution and by 
filtration of the inhibitor. The inactivation was partially prevented by pre-treatment of the enzyme with iodoacetamide. 
A slower simultaneous inactivation was observed which followed pseudo-first order kinetics. It is suggested that part of 
the rapid inhibition of CF, ATPase caused by DIMBOA is due to its reaction with sulthydryl groups on the enzyme. 
These reactions of DIMBOA may explain some of the inhibitory properties of DIMBOA on a wide range of organisms. 
INTRODUCTION 
Hydroxamic acids isolated from extracts of the 
Gramineae such as wheat, maize and rye [l] inhibit 
bacterial [2] and fungal [3] growth as well as insect 
development and reproduction [&7]. These hydroxamic 
acids have been suggested as resistance factors of maize 
against the European corn borer Ostrinia nubikzlis [4] and 
the aphid Rhopnlosiphum maidis [5], and of wheat against 
other aphids [6, 71. Recently DIMBOA (l), the main 
hydroxamic acid in maize extracts, was shown to inhibit 
energy transfer reactions [8]. The properties of DIMBOA 
as an energy transfer inhibitor may account for its 
widespread toxicity [8]. It was additionally shown that 
DIMBOA inhibits chloroplast coupling factor 1 (CF,). 
DIMBOA reacts with sulfhydryl compounds forming 
addition and/or reduction products [9, lo]. In this paper 
the inhibition by DIMBOA of the ATPase activity of CF, 
from spinach chloroplasts is further characterized. It is 
suggested that this inhibition is partly due to the reaction 
of DIMBOA with sulfhydryl groups on the enzyme. 
2 
RESULTS 
DIMBOA inhibited the ATPase activity of CF, from 
spinach chloroplasts. The inhibition was about 50 7 with 
5mM DIMBOA (Fig. 1 and Table 1). The reaction of 
$To whom correspondence should be addressed. 
DIMBOA with the enzyme was completed fast enough so 
that no further inhibition was appreciable during the 
assay period. A Hill plot of the inhibition of ATP 
hydrolysis by DIMBOA (Fig. 1, inset) gave an apparent n 
value of 1.03, suggesting that the reaction of 1 mol of 
hydroxamic acid per mol of active site is responsible for 
the enzyme inactivation. The inhibition was reversible 
since a 222-fold dilution of the inhibited enzyme or gel 
filtration restored the original activity (Table 1). This 
reversible inhibition was largely uncompetitive (Fig. 2), 
suggesting that the inhibitor binds mainly to the 
enzymeesubstrate complex at a place other than the 
substrate binding site. 
J 
0 
DIMBCI~ (mM) 
10 
Fig. 1. Inhibition of CF, ATPase by DIMBOA. Tubes contain- 
ing 1 ml of 40 mM Tricine--NaOH (pH 8), 5 mM ATP, 5 mM 
CaCI,, different concentrations of DIMBOA and 3pg of CF, 
were incubated at 37”. Inorganic phosphate was determined after 
7 min as described in Experimental. The activity of the control 
was 13.8 pmol Pi/mg protein per min. 
2455 
2456 C. B. QUEIROLO et al 
Table 1. Reversibility of the inhibition of CF, ATPase by 
DIMBOA with short pre-incubation times 
Treatment of 
samples 
DIMBOA Activity 
concentration (pm01 Pi/mg 
(mM) protein 
Initial* Final t per min) 
No filtration 0 0 9.6 
No filtration 0 0.02 9.4 
No filtration 5 5 4.8 
Filtration 0 0 9.6 
Filtration 5 0 9.6 
Inhibitor dilution 5 0.02 9.3 
ATP, CaCI, and DIMBOA were added to tubes containing 
activated CF, (89pg) in 100/11 of incubation medium (40mM 
tricine-NaOH, pH 8, and 2rhM EDTA) in order to reach 
concentrations of 5, 7 and 5mM, respectively. Similar control 
tubes without DIMBOA were set up. The tubes were incubated at 
25” for 5 min and then filtered through Sephadex G-50 using the 
rapid centrifugation-filtration technique of Penefsky [ 181. 
Protein determinations were carried out before and after filt- 
ration. ATPase activities before and after filtration were de- 
termined by transferring aliquots (4.5~1) to 1 ml of a reaction 
medium containing 40 mM Tricine-NaOH (pH 8), 2 mM EDTA, 
5 mM ATP and 7 mM CaCl,, and incubating for 8 min at 37”. In 
addition, activities before filtration were measured in the tube 
with inhibitor in a reaction medium containing 5 mM DIMBOA, 
and in the tube without inhibitor in a reaction medium contain- 
ing 0.02 mM DIMBOA (concentration after dilution in the 
reaction medium for the second incubation). 
‘DIMBOA concentration during the initial 5 min incubation. 
+DIMBOA concentration during the second incubation in 
which activity was measured. In some treatments the concentra- 
tion of DIMBOA was kept constant by adding the appropriate 
amount of the inhibitor after the dilution made for the second 
incubation. 
0 3 6 
CaATP lmM) 
Fig. 2. Hanes plots [20] of substrate dependence of the ATPase 
inhibition by DIMBOA. Tubes containing 1 ml of 40mM 
Tricine-NaOH (pH 8), different concentrations of CaATP and 
3.4 pg of CF, were incubated at 37” in the absence (0) or presence 
of 2 (0) or 5 mM (0) DIMBOA. Inorganic phosphate liberated 
was determined after 8 min as described. 
ADP inhibited the ATPase reaction, the saturation 
function with ATP becoming sigmoidal upon addition of 
the inhibitor [Ill]. The sigmoidicity was not altered by 
DIMBOA (Fig. 3), indicating that DIMBOA does not 
interfere appreciably with the allosteric site. Hill coef- 
ficients in the absence of ADP were 0.9 with DIMBOA 
and 1.1 without it, and in the presence of ADP were 1.6 
with DIMBOA and 1.8 without it 
0 
CoATi (mM) 
6 
Fig. 3. Effect of DIMBOA on CF, ATPase in the presence and 
absence of ADP. ATPase activity of CF, was measured in the 
presence of different concentrations of CaATP in 1 ml of 40 mM 
Tricine-NaOH (pH 8) (control, 0) and I mM CaADP (W) or 
5 mM DIMBOA (0) or 1 mM CaADPand 5 mM DIMBOA (0). 
The reaction was started with the addition of 1.5pg of CF, and 
was carried out for Smin at 37 Inorganic phosphate was 
measured as described. 
Since DIMBOA reacts with thiols [9, lo] and after 
activation CF, has four sulfhydryl groups [12, 131, the 
effect of iodoacetamide on the inhibition by DIMBOA 
was studied. Alkylation of the enzyme with iodoaceta- 
mide, although not inhibiting the enzyme significantly 
[ 121, appeared to protect the enzyme partially against the 
inhibition by DIMBOA (Fig. 4). The fact that protection 
of the enzyme by iodoacetamide was only partial suggests 
that cysteine residues are not the only groups reacting 
with DIMBOA to cause the observed inhibition. 
In addition to the fast reversible reaction between CF, 
and DIMBOA described above, a slow inhibition was 
observed (Fig. 5). This inhibition was recognized as 
irreversible because it persisted in spite of the near 300- 
fold dilution of the inhibitor during the assay. This 
inhibition followed pseudo-first order kinetics (Fig. 5), 
suggesting that it is a single homogeneous process, i.e. 
only one group on the enzyme reacted with DIMBOA, or 
more than one group with comparable reactivities to- 
wards DIMBOA are reacting. This process was not 
further characterized. 
DISCUSSION 
DIMBOA equilibrates rapidly in aquequs solutions 
with the open-chain compound 2. The aldehyde function 
of this species is a highly reactive electrophile since it has 
an adjacent carbonyl group draining electron density 
DIMBOA (mM) 
Fig. 4. Effect of treatment of CF, with iodoacetamide on the 
inhibition of CF, ATPase by DIMBOA. The time required for 
maximal protection by iodoacetamide from the inhibition by 
5 mM DIMBOA is shown in the inset. CF, (22Opg) was added to 
400~1 of incubation medium (40mM Tricine-NaOH, pH 8, 
2mM EDTA and 1OmM iodoacetamide) at 25”. After different 
away from it. Furthermore, the nitrogen atom in 2 is 
prone to attack by reducing agents due to electron 
withdrawal from the adjacent carbonyl groups. In the 
presence of nucleophiles DIMBOA should thus form 
addition products quickly and reversibly. In the presence 
of reducing agents the hydroxamic nitrogen atom might 
be slowly reduced to an amide nitrogen. Thiols are both 
good nucleophiles and good reducing agents, and the 
reactions described above have been shown to occur 
between DIMBOA and thiols in aqueous solutions at 
pH 8 [9]. It is conceivable then that the enzyme inactiv- 
ation caused by DIMBOA is partly due to its reaction 
with sulfhydryl groups in the enzyme. Sulfhydryl groups 
are normally more nucleophilic than amino and hydroxyl 
groups [14]. However, when the latter are present in an 
enzyme, they may also react with DIMBOA to cause 
additional inhibition. 
The experiments described above show that DIMBOA 
caused a fast and reversible inhibition of the enzyme. The 
inhibition by DIMBOA was partly prevented by alky- 
lation by iodoacetamide of sulfhydryl groups on the 
enzyme. In addition, the inhibition by DIMBOA was 
uncompetitive, consistent with the absence of cysteine 
residues in the substrate binding site. It is likely that 
reaction of DIMBOA with sulfhydryl groups on the 
enzyme induced a conformational change in the enzyme 
upon which it lost its normal activity. With longer reaction 
times between DIMBOA and the enzyme, an irreversible 
inhibition process appeared. The nature of this reaction 
remains to be clarified. 
time intervals, 60~1 aliquots were withdrawn and filtered using 
the technique of Penefsky [18]. The enzyme reaction was carried 
out by adding aliquots of the filtrate into 1 ml of 40mM 
Tricine-NaOH, pH 8, and 5 mM CaATP with and without added 
5mM DIMBOA, and after 5min. inorganic phosphate was 
determined as described. Each tube contained 4.2pg of CF, 
treated with iodoacetamide (0) or untreated CF, (0). A time 
equal to 90 min was chosen for studying the effect in the presence 
of other concentrations of DIMBOA. CF, (615pg in 800~1 of 
incubation medium) was allowed to react for 90min at 25” with 
and without 1OmM iodoacetamide. ATPase activities were then 
measured for S min in the presence of different concentrations of 
DIMBOA. 
199 
inhibitory in this and a previous study [8] suggest - 
compartmentation at tissue and/or cellular levels in such a 
way that they would not affect metabolism. 
EXPERIMENTAL 
Fig. 5. Kinetics of CF, ATPase inhibition by DIMBOA. Two 
test-tubes containing 1.3 pg/ml of CF,, 40 mM Tricine-NaOH 
(pH 8) and 2mM EDTA were kept at 25”. A solution of 
DIMBOA in dimethyl sulfoxide was added to one of them 
making the solution 5mM in DIMBOA. At different time 
intervals, an aliquot was withdrawn (3.5 ~1) and added to tubes 
containing 1 ml of the ATPase assay medium where the activity 
was measured as described for 8 min. Activity at time zero was 
measured without inhibitor and with 17.5 PM DIMBOA (con- 
centration after dilution in the assay medium), giving in both 
cases the same results. Activities of the controls were 7.8pmol 
x c 
0 -, 
0 70 1LO 
Time (mid 
Pi/mg protein per min. 0 = activity; 0 = log activity. 
Isolation of DIMBOA. The inhibitor was isolated by a 
modification of the method in ref. [15]. Seeds of Zea mays L. cv 
Tl29s were sown in humus, watered with tap water, and grown 
under continuous light in a greenhouse at 28 If: 2”. Leaves of 7- 
day-old seedlings were extracted and the macerate was filtered 
through cheesecloth and adjusted to pH 3 by the addition of 
0.1 M HCl. The acid extract was then centrifuged at 85OOg for 
1Omin and the supcrnatant partitioned against Et,0 (2 ~01s. x 3). 
The Et,0 phases were pooled and the solvent removed under 
vacuum. The crystalline residue was recrystallized from 
dioxane-petrol giving fine white needles which were character- 
ized as DIMBOA by UV, IR and ‘H NMR spectroscopy. 
isolation and purz$cation of CF,. Class II chloroplasts were 
isolated from market spinach leaves (Spinacia oleracea L.) as 
described in ref. [16]. Thylakoid membranes were subjected to 
low ionic strength and CF, was extracted with EDTA. CF, was 
purified by precipitation with (NH&SO4 and chromatography 
on DEAE-Sephadex, as described in ref. [17]. Electrophoresis on 
polyacrylamide gel showed it was at least 95 % pure. CF, was re- 
precipitated with (NH&SO4 and stored at 3”. 
Activation of ATPase. Activation was carried out as described 
in ref. r121. Aliauots of ourified CF, precipitated with L., . __ - 
Inhibition of ATPase from chloroplast by DIMBOA 2451 
DIMBOA is deleterious to a wide range of organisms. 
Its activity as an energy transduction inhibitor [8] may 
account for its widespread toxicity. However, since 
DIMBOA reacts with thiols, other enzymes may also be 
expected to be inhibited by this molecule. The naturally- 
occurring concentration of DIMBOA (as glycosides) and 
related benzoxazinones (up to 5 mmol/kg fresh weight) in 
the Gramineae [7] as compared with those shown to be 
2458 C. B. QUEIROLO et al. 
(NH&SO, were centrifuged for 3 min. The pellet was dissolved 
in a medium containing 40mM Tricine-NaOH (pH 8), 50ml 
dithioerythreitol and 2 mM EDTA. After incubating the mixture 
for 3 hr at 25”, it was filtered through Sephadex G-50equilibrated 
with 40mM Tricine-NaOH (pH 8) using the rapid 
centrifugation-filtration technique of ref. [18]. Protein determi- 
nations were carried out by the method of Lowry using bovine 
serum albumin as a standard. 
ATPase activity. After 5-8 min of the enzyme-catalysed reac- 
tion at 37”, the reaction mixtures (1 ml) containing 1.5%5pg of 
CF, were quenched with 1 ml of ice-cold trichloroacetic acid 
(TCA). Free inorganic phosphate was determined colorimetri- 
tally [19]. Blanks were made by delaying the addition of CF, 
until the aliquots of the reaction mixture had been mixed with 
TCA. The ATPase reaction was shown to be linear for at least 
1Omin at 37” with 5mM CaATP and for at least 8min with 
0.4 mM CaATP. 
Acknowledgements--We thank Drs. Hermann Niemeyer and 
Ruben H. Vallejos for their help and critical revision of this 
manuscript. This research was supported by the International 
Foundation for Science (grant 484), UNDPjUNESCO Program 
RLA 78/024, Universidad de Chile (grant B622) and Consejo 
National de Investigaciones Cientificas y Tecnicas, Argentina. 
REFERENCES 
1. Willard, J. I. and Penner, D. (1976) Residue Rev. 64, 67. 
2. Corcuera,,L. J., Woodward, M. D., Helgeson, J. P., Kelman, 
A. and Upper, C. D. (1978) Plant Physiol. 61, 791. 
3. ElNaghy, M. A. and Linko, P. (1962) Physiol. Plant. 15,764. 
4. Klun, J. A., Tipton, C. L. and Brindley, T. A. (1967) J. Econ. 
5. 
6. 
8. 
9. 
10. 
11. 
12. 
13. 
14. 
15. 
16. 
17. 
18. 
19. 
20. 
Entomol. 60, 1529. 
Long, B. J., Dunn, G. M., Bowman. J. S. and Routley, D. G. 
(1977) Crop sci. 17, 55. 
Argandofia, V. H., Luza, J. G., Niemeyer, H. M. and 
Corcuera, L. J. (1980) Phytochemislry 19, 1665. 
Argandofia, V. H., Niemeyer, H. M. and Corcuera, L. J. 
(1981) Photochemistry 20, 673. 
Queirolo, C. B., Andreo, C. S., Vallejos, R. H., Niemeyer. 
H. M. and Corcuera, L. J. (1981) Plant Physiol. 68, 941. 
Niemeyer, H. M., Corcuera, L. J. and PCrez, F. J. (1982) 
PhytochemistrJ 21, 2287. 
Argandofia, V. H., Peila, G. F., Niemeyer, H. M. and 
Corcuera. L. J. (1982) Phytochemistr_v 21, 1573. 
Nelson, N., Nelson, H. and Racker, E. (1972) .I. Biol. Chem. 
247, 6505. 
Arana, J. L. and Vallejos, R. H. (1982) J. Biol. C’hem. 257. 
1125. 
Vallejos, R. H. (1981) in Energy Coupling in Photosynthesis 
(Selman, B. R. and Selman-Reiner, S., eds.), pp. 129-139. 
ElsevierjNorth Holland, New York. 
Friedman, M. (1973) The Chemistry and Biochemistry 01 the 
Sulfhydryl Group in Amino Acids, Peptides and Proteins. 
Pergamon Press, Oxford. 
Woodward, M. D.. Corcuera, L. J., Helgeson, J. P. and 
Upper, C. D. (1978) Plan1 Phqasiol. 61. 796. 
Raviuini, R. A., Andreo, C. S. and Vallejos, R. H. (1977) Planr 
Cell Physiol. 18, 701. 
Lien, S. and Racker, E. (1971) Methods Enzymol. 23, 547. 
Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891. 
Taussky, H. and Shorr, E. (1953) J. Biol. Chem. 202, 675. 
Cornish-Bowden, A. (1976) Principles of Enzyme Kinerics. 
Butterworths. London, 


Inhibition of ATPase from chloroplasts by a hydroxamic acid from Gramineae.

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=4ACDA85C6E4A98721F17D2B879D11DAE?doi=10.1.1.1072.8410&rep=rep1&type=pdf

Inhibition of ATPase from chloroplasts by a hydroxamic acid from Gramineae.

Abstract

Similar works

Full text

Available Versions

CiteSeerX