Interaction of Herbicides with Cellular and Liposome Membranes

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Interaction of Herbicides with Cellular and Liposome Membranes DONALD E . MORELAND, STEVEN C. HUBER, and WILLIAM P. NOVITZKY North Carolina State University, U.S. Department of Agriculture, Agricultural Research Service, Departments of Crop Science and Botany, Raleigh, NC 27650

Many h e r b i c i d e s inhibit c h l o r o p l a s t e l e c t r o n t r a n s p o r t by b i n d i n g to a p r o t e i n l o c a t e d on the reducing s i d e of PS II. Some of the h e r b i c i d e s (chlorpropham, dinoseb, p r o p a n i l , ioxynil), but not all (diuron, s-triazines, uracils), also interfere w i t h photophosphorylation and m i t o c h o n d r i a l e l e c t r o n transport and p h o s p h o r y l a t i o n . I n t h i s study, p r o p a n i l , dinoseb, ioxynil, and s e v e r a l c a r b a n i l a t e s , but not d i u r o n , a f f e c t e d the f o l l o w i n g responses, much like the uncoupler FCCP: (a) inhibited the light-dependent quenching of a t e b r i n fluorescence in t h y l a k o i d s ; (b) inhibited v a l i n o m y c i n -induced s w e l l i n g of all o r g a n e l l e s ; (c) increased the p e r m e a b i l i t y of all o r g a n e l l e membranes to K+ in the absence of an ionophore; and (d) increased the p e r m e a b i l i t y of p h o s p h a t i d y l c h o l i n e liposomes to H. The structure/activity c o r r e l a t i o n s were similar f o r all o r g a n e l l e s , i.e., the responses were not membrane specific. R e s u l t s obtained suggested that when h e r b i c i d e s partition i n t o the lipid phases of o r g a n e l l e membranes, p e r t u r b a t i o n s are produced that l e a d to a l t e r a t i o n s in "fluidity" and p e r m e a b i l i t y to c a t i o n s . The a l t e r a t i o n s may be r e s p o n s i b l e f o r uncoupling of ATP generation i n mitochondria and c h l o r o p l a s t s , and inhibition of e l e c t r o n t r a n s p o r t in m i t o c h o n d r i a . +

A l a r g e number of commercial h e r b i c i d e s i n t e r f e r e with e l e c t r o n transport and ATP production i n i s o l a t e d c h l o r o p l a s t s and mitochondria (1). These h e r b i c i d e s can be d i v i d e d i n t o two groups: e l e c t r o n transport i n h i b i t o r s and i n h i b i t o r y uncouplers (1, 2 ) . The dimethylphenylureas, s u b s t i t u t e d u r a c i l s , s ~ t r i a z i n e s , and pyridazinones have been c l a s s i f i e d as e l e c t r o n

This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

Moreland et al.; Biochemical Responses Induced by Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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BIOCHEMICAL RESPONSES INDUCED BY HERBICIDES

t r a n s p o r t i n h i b i t o r s , whereas the a l k y l a t e d d i n i t r o p h e n o l s , a c y l a n i l i d e s , halogenated b e n z o n i t r i l e s , and #-phenylcarbamates have been c l a s s i f i e d as i n h i b i t o r y uncouplers. A l l of the abovenamed h e r b i c i d e s i n h i b i t c h i o r o p l a s t e l e c t r o n t r a n s p o r t by b i n d i n g r e v e r s i b l y to a p r o t e i n ( s ) a s s o c i a t e d w i t h the Β complex (3, ^ , 5 ) . Β i s considered to f u n c t i o n as the secondary acceptor of e l e c t r o n s from PS I I . Binding a f f i n i t y of the h e r b i c i d a l i n h i b i t o r s has been c o r r e l a t e d w i t h i n h i b i t i o n of PS I I . A d d i t i o n a l d e t a i l s of the b i n d i n g responses of the h e r b i c i d e s and chemical models proposed to e x p l a i n the i n t e r a c t i o n between i n h i b i t o r s and the receptor t a r g e t are provided i n other c o n t r i b u t i o n s published h e r e i n C5, 6^, 7) · The e l e c t r o n transport i n h i b i t o r s do not d i r e c t l y a f f e c t photophosphorylation or i n t e r f e r e w i t h m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and phosphorylation (1) . However, the i n h i b i t o r y un c o u p l e r s , i n a d d i t i o n to i n t e r f e r i n g w i t h e l e c t r o n t r a n s p o r t i n t h y l a k o i d s , uncouple photophosphorylation and o x i d a t i v e phosphory l a t i o n , and i n h i b i t m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t . Whereas i n h i b i t i o n of c h i o r o p l a s t e l e c t r o n transport has been c o r r e l a t e d with b i n d i n g to a p r o t e i n ( s ) , the s i t e s and mechanisms through which h e r b i c i d e s i n t e r f e r e w i t h m i t o c h o n d r i a l and c h i o r o p l a s t mediated phosphorylations remain to be i d e n t i f i e d . When l i p o p h i l i c h e r b i c i d e s p a r t i t i o n i n t o the l i p i d phases of mem branes, they could perturb l i p i d - l i p i d , l i p i d - p r o t e i n , and p r o t e i n - p r o t e i n i n t e r a c t i o n s that are r e q u i r e d f o r membrane func t i o n s such as e l e c t r o n t r a n s p o r t , ATP formation, and a c t i v e t r a n s p o r t . Evidence f o r general membrane p e r t u r b a t i o n s caused by chlorpropham, 2 , 6 - d i n i t r o a n i l i n e s , p e r f l u i d o n e , and c e r t a i n phenylureas have been reported p r e v i o u s l y (8-11). The o b j e c t i v e s of the s t u d i e s reported here were to compare the a c t i o n of the compounds i d e n t i f i e d i n Table I (a) on c h i o r o p l a s t and m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and phosphorylation, and (b) on the " f l u i d i t y " and p e r m e a b i l i t y to KT and H+ of c h i o r o p l a s t , m i t o c h o n d r i a l , and liposome membranes. For com p a r a t i v e purposes, dinoseb, i o x y n i l , p r o p a n i l , and 3-CIPC (chlorpropham) were i n c l u d e d to represent the i n h i b i t o r y un c o u p l e r s . Diuron represented the e l e c t r o n t r a n s p o r t i n h i b i t o r s , and FCCP was i n c l u d e d as a reference uncoupler. Three other c a r b a n i l a t e s (3-CHPC; 2,3-DCIPC; and 3,4-DCIPC) were s e l e c t e d to determine the e f f e c t of replacement of the i s o p r o p y l s i d e chain with a hexyl moiety and the e f f e c t of d i c h l o r i n a t i o n of the phenyl r i n g i n the 2,3- and 3 , 4 - p o s i t i o n s . In the phenylureas and a c y l a n i l i d e s , i n c r e a s i n g the length of the s i d e chain has been a s s o c i a t e d w i t h increased i n h i b i t o r y a c t i v i t y against the H i l l r e a c t i o n (12). A l s o , i n the phenylureas, phenylcarbamates, and a c y l a n i l i d e s , d i c h l o r i n a t i o n i n the 3,4-ring p o s i t i o n s i s more i n h i b i t o r y to c h i o r o p l a s t e l e c t r o n t r a n s p o r t than monochlorin a t i o n i n e i t h e r p o s i t i o n . A d d i t i o n a l l y , c h l o r i n a t i o n i n an ortho p o s i t i o n has been a s s o c i a t e d w i t h decreased i n h i b i t o r y a c t i v i t y (12).


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TABLE I Common names or d e s i g n a t i o n s and chemical names of compounds s t u d i e d .

Common name or d e s i g n a t i o n

Chemical name

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2.3- DCIPC 3.4- DCIPC

carbonylcyanide p-trifluromethoxyphenylhydrazone 2-S£c-butyl-4,6-dinitrophenol 3-(3,4-dichlorophenyl)-1,1-dimethylurea 4-hydroxy-3,5-diiodobenzonitrile 3 ,4'-dichloropropionanilide isopropyl m-chlorocarbanilate hexyl m-chlorocarbanilate isopropyl 2,3-dichlorocarbanilate isopropyl 3,4-dichlorocarbanilate 1

M a t e r i a l s and Methods Chloroplasts. I n t a c t c h l o r o p l a s t s were i s o l a t e d from f r e s h l y harvested growth chamber-grown spinach (Spinaoia oleraoea L.) as described by L i l l e y and Walker (13). Thylakoids were prepared by the method of Armond et al. (14). C h l o r o p h y l l concentrations were determined by the method of MacKinney (15). Reaction cuvettes were i l l u m i n a t e d at 25 C with a photon f l u e n c e r a t e of 7.5 X 10~^ mol/m^-s (PAR). Reduction of f e r r i c y a n i d e with water as the oxidant was measured s p e c t r o p h o t o m e t r i c a l l y at 420 nm i n a r e a c t i o n medium (5.0 ml volume) that contained 0.1 M s o r b i t o l , 50 mM tricine-NaOH (pH 8.0), 0.4 mM KH2PO4, 5 mM M g C l , 10 mM NaCl, 0.5 mM K Fe(CN)6, 1 mM ADP, and t h y l a k o i d s (100 yg c h l o r o p h y l l ) . E s t e r i f i c a t i o n of i n o r g a n i c phosphate was measured by the procedure of L a n z e t t a et al. (16). E f f e c t s on the l i g h t dependent quenching of a t e b r i n were measured i n a r e a c t i o n medium (2.0 ml volume) that contained 1.0 yM a t e b r i n , 0.1 M s o r b i t o l , 10 mM tricine-NaOH (pH 7.8), 10 mM NaCl, and t h y l a k o i d s (20 yg c h l o r o p h y l l ) . The t h y l a k o i d s were i l l u m i n a t e d w i t h red a c t i n i c l i g h t (Corning 2-64 f i l t e r ) . The a t e b r i n was e x c i t e d with 366 nm l i g h t and emission was observed at 505 nm. E f f e c t s imposed on the e f f l u x of K were measured by suspending i n t a c t c h l o r o p l a s t s (100 yg c h l o r o p h y l l ) i n a r e a c t i o n medium (1.0 ml volume) that contained 0.4 M s o r b i t o l , and 10 mM HepesNaOH (pH 7.1). A f t e r i n c u b a t i o n with t e s t chemicals f o r 2 min, the r e a c t i o n mixtures were c e n t r i f u g e d to p e l l e t the c h l o r o p l a s t s . id" content of the supernatants was measured by flame photometry. 2

3

+

Mitochondria. Mitochondria were prepared from 3-day-old dark-grown mung bean (Phaseolus aureus Roxb.) h y p o c o t y l s . The i s o l a t i o n procedure, measurements of oxygen u t i l i z a t i o n , and


82


e f f e c t s of the t e s t compounds on r e s p i r a t o r y s t a t e s were conducted as described p r e v i o u s l y ( 9 ) . Following the terminology of Chance and Williams (17), the ADP-stimulated r a t e of r e s p i r a t i o n w i l l be r e f e r r e d to as s t a t e 3 and ADP-limited r e s p i r a t i o n as s t a t e 4. The mung bean mitochondria had r e s p i r a t o r y c o n t r o l ( s t a t e 3/state 4) r a t i o s that averaged 3.9, 3.6, and 2.2; and c a l c u l a t e d ADP/O r a t i o s that averaged 2.3, 1.3, and 1.5, f o r the o x i d a t i o n of malate, NADH, and s u c c i n a t e , r e s p e c t i v e l y . Osmotic S w e l l i n g . Changes i n the osmotic s t a b i l i t y of the o r g a n e l l e s were monitored s p e c t r o p h o t o m e t r i c a l l y at 550 nm f o r c h l o r o p l a s t s and t h y l a k o i d s , and at 520 nm f o r mitochondria. The 2.0-ml r e a c t i o n mixture contained 0.15 M KSCN or KC1 and 10 mM Hepes-NaOH (pH 7.1). The i n i t i a l absorbance of the r e a c t i o n was adjusted to 0.8, 0.4, and 0.75 f o r c h l o r o p l a s t s (approximately 20 yg c h l o r o p h y l l ) , t h y l a k o i d s (approximately 20 yg c h l o r o p h y l l ) , and mitochondria (approximately 0.4 mg p r o t e i n ) , r e s p e c t i v e l y . Induction of p a s s i v e s w e l l i n g was measured with the t e s t compound being added 30 s a f t e r the i n t r o d u c t i o n of the o r g a n e l l e s . In s t u d i e s with valinomycin (0.1 yM), the t e s t compound was added 30 s p r i o r to the i n t r o d u c t i o n of the ionophore. Rates of s w e l l i n g were c a l c u l a t e d from the i n i t i a l phase of absorbance decrease. Liposomes. Liposomes were prepared by s o n i c a t i o n from egg y o l k phosphatidyl c h o l i n e (Sigma type X-E) according to the method of H i n k l e (18). The assay medium used to determine e f f e c t s of h e r b i c i d e s and FCCP contained 0.2 ml liposomes i n 1.8 ml of 0.3 M NaCl, 20 mM t r i s - H C l (pH 7.5), 5 mM Na-ascorbate, 80 yM ferrocene, and 80 yM tetraphenylboron. F e r r i c y a n i d e r e d u c t i o n was measured s p e c t r o p h o t o m e t r i c a l l y at 420 nm and 25 C. Test Chemicals. Stock s o l u t i o n s of the d e s i r e d concentrat i o n s of t e s t chemicals were prepared i n acetone. The f i n a l concentration of the s o l v e n t was h e l d constant at 1% (v/v) i n a l l assays i n c l u d i n g the c o n t r o l s . Data presented f o r the s e v e r a l s t u d i e s were averaged from determinations made w i t h a minimum of three separate r e p l i c a t i o n s and i s o l a t i o n s . Results

and

Discussion

Shown i n Table I I are I^Q values f o r i n h i b i t i o n of e l e c t r o n t r a n s p o r t i n spinach t h y l a k o i d s (water to f e r r i c y a n i d e ) and the a s s o c i a t e d phosphorylation r e a c t i o n by the t e s t compounds. Also i n c l u d e d are I 5 0 values f o r i n h i b i t i o n of the light-dependent quenching of a t e b r i n f l u o r e s c e n c e and the r a t i o obtained by d i v i d i n g the I C Q f o r the a t e b r i n response by the I 5 Q f o r i n h i b i t i o n of photophosphorylation. The r e l a t i v e order of i n h i b i t o r y potency f o r some of the compounds f o r i n h i b i t i o n of f e r r i c y a n i d e r e d u c t i o n has been reported p r e v i o u s l y (12). For the h e r b i c i d e s ,


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TABLE I I E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on r e a c t i o n s mediated by spinach t h y l a k o i d s .

Ferricyanide PhosphoryReduction lation

Compound

•I

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC

7

NE^ 7.5 0.07 0.3 0.7 150 62 130 12

5 0

Atebrin fluor.

Atebrin/ phosph. ratio

(PM)

0.35 4 0.08 0.2 0.6 140 15 125 8

0.4 23 NE 120 200 130 28 NE 70

— NE = no, or minimal, e f f e c t w i t h concentrations

1 6 —

600 333 1 2 —

9

up to 400 yM.

except diuron (a pure e l e c t r o n t r a n s p o r t i n h i b i t o r ) , the I 5 0 f o r i n h i b i t i o n of the coupled phosphorylation was lower than the I 5 0 f o r i n h i b i t i o n of f e r r i c y a n i d e r e d u c t i o n . A lower I^Q value f o r i n h i b i t i o n o f phosphorylation suggests that the compounds might be expressing an e f f e c t on the phosphorylation pathway that cannot be explained e n t i r e l y by i n t e r f e r e n c e w i t h e l e c t r o n t r a n s p o r t . The h e r b i c i d e s , except f o r d i u r o n , have been shown p r e v i o u s l y to i n h i b i t c y c l i c photophosphorylation measured i n the absence of oxygen (1), and the l i g h t - i n d u c e d s y n t h e s i s of ATP mediated by d i t h i o t h r e i t o l and PMS (19). The l a t t e r r e a c t i o n s do not i n v o l v e the e n t i r e e l e c t r o n t r a n s p o r t chain and are i n s e n s i t i v e to diuron. An i n c r e a s e i n l i p o p h i l i c i t y of 3-CIPC w i t h the replacement of a h e x y l f o r the i s o p r o p y l s i d e chain (3-CHPC versus 3-CIPC) r e s u l t e d i n increased i n h i b i t i o n of e l e c t r o n t r a n s p o r t and an even g r e a t e r i n h i b i t i o n of the coupled phosphorylation (Table I I ) . Increased r e d u c t i v e i n h i b i t o r y potency of 3,4-DCIPC over 3-CIPC a l s o i s shown. The i n h i b i t o r y uncouplers and the uncoupler FCCP, but not diuron and 2,3-DCIPC, a l s o i n h i b i t e d the l i g h t dependent quenching of a t e b r i n f l u o r e s c e n c e i n spinach t h y l a k o i d s (Table I I ) . I n h i b i t i o n of the light-dependent quenching of a t e b r i n fluorescence has been a t t r i b u t e d to d i s s i p a t i o n o f the energized s t a t e (Δ pH) of the t h y l a k o i d membrane, which i s considered to provide the d r i v i n g f o r c e f o r phosphorylation (20, 21). The i n t e r f e r e n c e s observed support the suggestion that the i n h i b i t o r y uncoupler


84


h e r b i c i d e s do act as uncouplers. I n h i b i t i o n of l i g h t - i n d u c e d a t e b r i n fluorescence quenching by the c a r b a n i l a t e d e r i v a t i v e s a l s o was strengthened by an i n c r e a s e i n s i d e chain l i p o p h i l i c i t y (3-CIPC versus 3-CHPC) and d i c h l o r i n a t i o n (3-CIPC versus 3,4DCIPC), but negated by ortho s u b s t i t u t i o n (2,3-DCIPC). Of the p h e n o l i c h e r b i c i d e s , dinoseb was a good uncoupler, whereas i o x y n i l was a r e l a t i v e l y poor uncoupler. The i n h i b i t o r y un c o u p l e r s , but not the e l e c t r o n transport i n h i b i t o r s , a l s o stimulated e l e c t r o n t r a n s p o r t through PS I by i l l u m i n a t e d thy l a k o i d s (reduced DPIP as oxidant and methyl v i o l o g e n as reductant}, when e l e c t r o n flow through PS I I was blocked w i t h diuron (22). S t i m u l a t i o n o f oxygen uptake (22) and i n h i b i t i o n of the a t e b r i n induced fluorescence quenching (Table II) occurred over the same molar concentra t i o n ranges. Neither r e a c t i o n i n o v l v e d PS I I . The a t e b r i n / p h o s p h o r y l a t i o n r a t i o (Table I I , l a s t column) r e l a t e s i n h i b i t i o n of photophosphorylation to d i s s i p a t i o n of the p o s t u l a t e d energized s t a t e (Δ pH) o f the t h y l a k o i d membrane. A low r a t i o suggests that the two responses are c o r r e l a t e d . How ever, the high r a t i o s obtained f o r i o x y n i l and p r o p a n i l suggest that these two h e r b i c i d e s act as e l e c t r o n t r a n s p o r t i n h i b i t o r s r a t h e r than as uncouplers. M i t o c h o n d r i a l Responses. The h e r b i c i d e s r e f e r r e d to as i n h i b i t o r y uncouplers were so named because a t low molar concentra t i o n s they s a t i s f y most, i f not a l l , o f the c r i t e r i a e s t a b l i s h e d f o r uncouplers o f o x i d a t i v e p h o s p h o r y l a t i o n . However, a t h i g h e r molar c o n c e n t r a t i o n s , they a l s o i n h i b i t m i t o c h o n d r i a l e l e c t r o n transport ( 1 ) . Uncoupling a c t i o n ( i n t e r f e r e n c e w i t h ATP generation) has been demonstrated by s t i m u l a t i o n o f s t a t e 4 r e s p i r a t i o n f o r the o x i d a t i o n of malate, s u c c i n a t e , and NADH; circumvention of o l i g o m y c i n - i n h i b i t e d s t a t e 3 r e s p i r a t i o n ; and i n d u c t i o n of ATPase a c t i v i t y (1). In a d d i t i o n , by using v a r i o u s s u b s t r a t e s , p a r t i a l r e a c t i o n s , and e l e c t r o n mediators, evidence has been presented that the h e r b i c i d e s i n h i b i t malate o x i d a t i o n and malate-PMS oxidoreductase by a c t i n g a t o r near complex I , succinate o x i d a t i o n and succinate-PMS oxidoreductase by a c t i n g at o r near complex I I , exogenous-NADH o x i d a t i o n by a c t i n g p r i o r t o the cytochrome chain, and c y a n i d e - r e s i s t a n t r e s p i r a t i o n ( a l t e r n a t e o x i d a s e ) . That i n h i b i t i o n of malate, s u c c i n a t e , and exogenous-NADH o x i d a t i o n i s not caused by i n t e r f e r e n c e a t a common s i t e , shared by the 3 sub s t r a t e s , i s i n d i c a t e d by the widely d i f f e r i n g I 5 0 values that a r e obtained ( 1 , J3, 9_> 1 0 ) . The pure e l e c t r o n transport i n h i b i t o r s of c h i o r o p l a s t e l e c t r o n t r a n s p o r t have only a m a r g i n a l , i f any, e f f e c t on m i t o c h o n d r i a l responses. For the most p a r t , w i t h the compounds included i n t h i s study, maximum uncoupling a c t i v i t y ( s t i m u l a t i o n o f s t a t e 4 r e s p i r a t i o n ) was obtained f o r the o x i d a t i o n o f succinate and i n h i b i t i o n of malate s t a t e 3 r e s p i r a t i o n was most s e n s i t i v e . Therefore, only data f o r s t a t e 4 s t i m u l a t i o n f o r the o x i d a t i o n of succinate and


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TABLE I I I E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on r e a c t i o n s mediated by mung bean mitochondria.

Compound

Succinate state 4 respiration (Concn.) (Stim.) μΜ


—

NE = no,

0.1 1 , NR§/ 10 200 200 20 NE 40

Malate state 3 respiration (Inhib.)

% 108 92 NE 90 80 71 71 NE 69

or marginal, e f f e c t with concentrations

I (uM) 50

50 55 410 150 170 100 28 90 47

up

to 400

μΜ.

i n h i b i t i o n of s t a t e 3 r e s p i r a t i o n f o r the o x i d a t i o n of malate are presented i n Table I I I . Only diuron and 2,3-DCIPC f a i l e d to stimulate s t a t e 4 r e s p i r a t i o n . None of the compounds was as e f f e c t i v e an uncoupler as FCCP. P r o p a n i l and 3-CIPC showed maxi mum s t i m u l a t o r y a c t i v i t y at a r e l a t i v e l y high molar concentration (200 μ Μ ) . Replacement of the i s o p r o p y l group of CIPC f o r the h e x y l group (3-CHPC) was a s s o c i a t e d with an increase i n uncou p l i n g a c t i v i t y as was the a d d i t i o n of a second c h l o r i n e i n the 4 - p o s i t i o n (3,4-DCIPC) of the r i n g . For some of the h e r b i c i d e s , s t i m u l a t i o n of s t a t e 4 r e s p i r a t i o n occurred at molar concentra t i o n s that a l s o i n h i b i t e d s t a t e 3 r e s p i r a t i o n , which can be a t t r i b u t e d to an e f f e c t on e l e c t r o n t r a n s p o r t . Hence, the e f f e c t on e l e c t r o n transport may have masked the f u l l expression of uncoupling a c t i v i t y . A l l of the compounds i n h i b i t e d s t a t e 3 r e s p i r a t i o n with malate as s u b s t r a t e , however, diuron was a r e l a t i v e l y weak i n h i b i t o r . As i n the expression of uncoupling a c t i o n , the l e n g t h ening of the s i d e chain (3-CHPC versus 3-CIPC) and the a d d i t i o n of a second c h l o r i n e to the phenyl r i n g i n the para p o s i t i o n (3,4-DCIPC versus 3-CIPC) was a s s o c i a t e d w i t h enhanced a c t i v i t y . The 2,3-DCIPC d e r i v a t i v e was s l i g h t l y l e s s a c t i v e than 3-CIPC, the reference c a r b a n i l a t e . Whereas i n h i b i t i o n of c h i o r o p l a s t e l e c t r o n transport has been c o r r e l a t e d with b i n d i n g to a p r o t e i n ( s ) , the mechanisms f o r


86


i n t e r f e r e n c e w i t h photophosphorylation and the m i t o c h o n d r i a l responses remain to be i d e n t i f i e d . The h e r b i c i d e s could act independently with s e v e r a l components of the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t and p h o s p h o r y l a t i o n pathway, or they could be p e r t u r b i n g the membranes i n such a way that m u l t i s i t e i n t e r f e r e n c e results. Swelling Responses. E f f e c t s of the h e r b i c i d e s on the " f l u i d i t y " and p e r m e a b i l i t y p r o p e r t i e s of membranes were measured. A l t e r a t i o n s to the f l u i d i t y and p e r m e a b i l i t y of o r g a n e l l e membranes can a l t e r the osmotic p r o p e r t i e s of the membranes. C h i o r o p l a s t , t h y l a k o i d , and m i t o c h o n d r i a l membranes are known to be r e l a t i v e l y impermeable to c a t i o n s such as K*~ and but f r e e l y permeable to l i p o p h i l i c anions such as SCN"" (23, 24). Hence, o r g a n e l l e s are o s m o t i c a l l y s t a b l e when suspended i n i s o t o n i c KSCN. K p e r m e a b i l i t y , however, can be induced a r t i f i c i a l l y by ionophores such as valinomycin (Figure 1A). Valinomycin i s considered to form a l i p i d - s o l u b l e complex with ΚΓ*" and func t i o n s to t r a n s p o r t K across the membranes (25). An i n c r e a s e i n i n t e r n a l K+ w i l l be accompanied by the d i f f u s i o n of SCN", the counter i o n , across the membrane to maintain e l e c t r o n e u t r a l i t y . An i n c r e a s e i n i n t e r n a l s o l u t e c o n c e n t r a t i o n w i l l r e s u l t i n an i n f l u x of water and the o r g a n e l l e s w i l l s w e l l . Shown i n the lowest t r a c e s of F i g u r e IB, C, and D i s the r a t e and magnitude of s w e l l i n g obtained when valinomycin was added to i n t a c t c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria, r e s p e c t i v e l y . A l l of the t e s t compounds except d i u r o n , i n h i b i t e d the r a t e and magnitude of valinomycin-induced s w e l l i n g i n the three o r g a n e l l e s suspended i n i s o t o n i c KSCN, as shown i n F i g u r e 1 f o r dinoseb. Dose/response curves were developed from the t r a c e s (Figure 1) and I^Q values obtained from the curves are presented i n Table IV. The e f f e c t was expressed at molar concentrations higher than those r e q u i r e d to i n h i b i t c h i o r o p l a s t e l e c t r o n t r a n s port and p h o s p h o r y l a t i o n . However, the data demonstrate that i n h i b i t o r y uncouplers can a f f e c t the p r o p e r t i e s of o r g a n e l l e mem branes. The I 5 0 values f o r i n h i b i t i o n of valinomycin-induced s w e l l i n g i n mitochondria, w i t h the exception of FCCP, c o r r e l a t e d c l o s e l y with I 5 0 values f o r i n h i b i t i o n of malate s t a t e 3 r e s p i r a t i o n ( c f . Table I I I ) . Two of the c a r b a n i l a t e s (3,4-DCIPC and 3-CHPC) were as i n h i b i t o r y , or were b e t t e r i n h i b i t o r s , as i n d i cated by the lower I^Q v a l u e s , than the standard uncoupler FCCP, i n a l l three o r g a n e l l e s . +

+

The osmoticum used had a marked e f f e c t on the i n h i b i t o r y potency of FCCP and the non-carbanilates (Table I V ) . For the nonc a r b a n i l a t e h e r b i c i d e s and FCCP, valinomycin-induced s w e l l i n g of mitochondria suspended i n i s o t o n i c KC1 was i n h i b i t e d at much lower concentrations of the h e r b i c i d e s than when the mitochondria were suspended i n i s o t o n i c KSCN. For the c a r b a n i l a t e s , the molar c o n c e n t r a t i o n f o r 50% i n h i b i t i o n of valinomycin-induced s w e l l i n g of mitochondria suspended i n KSCN was about twice the


5.


MORELAND ET A L .

@

©

MODEL

87

THYLAKOIDS

MEMBRANE • HjO

DINOSEB

•SCN

μ -

INTACT CHLOROPLASTS DINOSEB VAL

® MITOCHONDRIA DINOSEB

I—0.5 min-

Figure 1. Representative traces of absorbance changes that show inhibition by dinoseb of valinomycin-induced swelling of intact spinach chloroplasts (B), spinach thylakoids (C), and mung bean mitochondria (D) suspended in isotonic KSCN. The model system is presented diagrammatically in A. Swelling was initiated by the addition of 0.1 Μ valinomycin (VAL). μ



88

TABLE IV I n h i b i t i o n of valinomycin-induced s w e l l i n g of i n t a c t spinach c h l o r o p l a s t s , spinach t h y l a k o i d s , and mung bean mitochondria by FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s .

Osmoticum Compound Chloroplasts

KSCN Thylakoids

Mitochondria

KCl Mitochondria

I (uM) 50

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2,3-DCIPC 3,4-DCIPC a/ — NE = no,

19 220 NEâ' 310 185 180 3 120 28

or minimal,

65 170 NE 240 180 135 14 95 17

0.1 0.4 300 2 5 100 14 66 25

22 90 NE 330 200 185 33 170 33

e f f e c t w i t h concentrations

up to 400

μΜ.

concentration r e q u i r e d to i n h i b i t the response w i t h mitochondria suspended i n K C l . I n h i b i t i o n of valinomycin-induced s w e l l i n g would occur i f the t e s t compounds i n t e r f e r e d w i t h movement of e i t h e r K* or anion, or both. Because valinomycin i s a mobile c a r r i e r of i t s r a t e of movement i s dependent upon the f l u i d i t y of the membrane. One i n t e r p r e t a t i o n f o r the observed i n h i b i t i o n s i s that the compounds, by p a r t i t i o n i n g i n t o the o r g a n e l l e membranes, decrease the " f l u i d i t y " of the membrane, and, subsequently, the r a t e at which the v a l i n o m y c i n - K complex moves across i t . The g r e a t e r s e n s i t i v i t y expressed w i t h KCl as the osmoticum suggested that FCCP and the i n h i b i t o r y uncouplers might a l s o i n t e r f e r e , i n some way, with CI" movement. The p o s s i b i l i t y e x i s t s t h a t , i n mung bean mito chondria, movement of C l ~ i s c a r r i e r - m e d i a t e d , whereas SCN", being a l i p o p h i l i c anion, d i f f u s e s f r e e l y across the membrane. The h e r b i c i d e s may, i n some manner, i n t e r f e r e with the endogenous t r a n s p o r t mechanism and, t h e r e f o r e , cause greater i n h i b i t i o n of swelling i n i s o t o n i c KCl. +

Not a l l endogenous membrane t r a n s p o r t systems are a f f e c t e d by the h e r b i c i d e s . For example, spontaneous m i t o c h o n d r i a l s w e l l i n g i n i s o t o n i c s o l u t i o n s of ammonium phosphate or n e u t r a l amino acids, was a f f e c t e d only m a r g i n a l l y by the compounds (data not shown). Swelling i n these systems i n v o l v e s the endogenous Pi"/0H~ a n t i p o r t e r (26) and amino a c i d p o r t e r (27) , r e s p e c t i v e l y . At t h i s


5.

MORELAND ET AL.


time, there i s no ready e x p l a n a t i o n f o r the apparent of C I " t r a n s p o r t by the n o n - c a r b a n i l a t e s .

89 inhibition

E f f e c t s on membrane p e r m e a b i l i t y to i o n s . For the most p a r t , the compounds induced c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria suspended i n i s o t o n i c KSCN t o s w e l l i n the absence of an ionophore. T y p i c a l r e s u l t s obtained w i t h dinoseb are presented i n F i g u r e 2. As shown i n the no-dinoseb c o n t r o l curves, some spontaneous s w e l l i n g occurred at a slow r a t e . However, the a d d i t i o n of dinoseb g r e a t l y i n c r e a s e d the r a t e and magnitude of the s w e l l i n g response i n a l l three o r g a n e l l e s . In these experiments, as i n the valinomycin s t u d i e s , s w e l l i n g would be expected to occur only i f the p e r m e a b i l i t y of the o r g a n e l l e membranes to was i n c r e a s e d by the t e s t compounds because the membranes are considered to be f r e e l y permeable to SCN" ( F i g u r e 2A). Dose/response curves were developed from t r a c e s such as those shown i n F i g u r e 2 f o r dinoseb. For comparative purposes, the conc e n t r a t i o n of compound r e q u i r e d to induce an i n c r e a s e i n the s w e l l i n g r a t e of 0.02 A i n 1 min r e l a t i v e to the n o - h e r b i c i d e cont r o l s i s shown i n Table V f o r c h l o r o p l a s t s , t h y l a k o i d s , and mitochondria. Thylakoids d i d not s w e l l as e x t e n s i v e l y as the o t h e r two o r g a n e l l e s , consequently, the values are f o r a change of 0.01 A. The r e l a t i v e order o f a c t i v i t y shown by the compounds i s s i m i l a r to that given i n Table IV f o r i n h i b i t i o n of v a l i n o m y c i n induced s w e l l i n g i n the three o r g a n e l l e s . Mitochondria suspended i n i s o t o n i c K C l were a l s o induced to s w e l l by a l l o f the compounds, except d i u r o n (Table V ) . However, as opposed to i n h i b i t i o n of valinomycin-induced s w e l l i n g (Table IV), the values obtained i n i s o t o n i c K C l were s l i g h t l y h i g h e r than those obtained i n i s o t o n i c KSCN, w i t h two exceptions (3-CHPC and 2,3-DCIPC). As i n the valinomycin-induced system, i n order f o r s w e l l i n g to occur, both the c a t i o n and anion must cross the membranes. Because SCN" i s a permeant anion and i t s movement i s c o n t r o l l e d e l e c t r o g e n i c a l l y , e f f e c t s imposed by the compounds on the movement of id" would determine the occurrence and extent of s w e l l i n g . Hence, with mitochondria suspended i n KSCN, data r e f l e c t e f f e c t s imposed on the movement of id". In mitochondria suspended i n K C l , the compounds could a f f e c t movement of e i t h e r or both id" and C l ~ because both are nonpermeant. I f the compounds were i n c r e a s i n g only the p e r m e a b i l i t y of the m i t o c h o n d r i a l membrane to K", then data obtained i n KSCN and K C l would be expected to be s i m i l a r . The higher values obtained i n K C l r e l a t i v e t o KSCN may r e f l e c t i n t e r f e r e n c e by the compounds with C l ~ movement. As f o r i n h i b i t i o n o f valinomycin-induced s w e l l i n g , some o f the c a r b a n i l a t e s (3-CHPC and 2,3-DCIPC) seem to be a c t i n g d i f f e r e n t l y i n t h a t values obtained i n i s o t o n i c K C l were lower than those obtained i n i s o t o n i c KSCN. The p e r m e a b i l i t y of c h i o r o p l a s t membranes to endogenous K+ was a l s o i n c r e a s e d by FCCP and the i n h i b i t o r y uncouplers, but not 4


90


(§)

MODEL

©

THYLAKOIDS

MEMBRANE OUT/ SCN"

/

I/ /

IN

DINOSEB

• SCN"

HoO

INTACT CHLOROPLASTS

τ

0.05A

1 J-

M 0

®

MITOCHONDRIA

DINOSEB

DINOSEB

V

\ \ \

^°

V \ΝΛιοο \V ^ 2 0 0 \ 400 1—

1 min —

-0.5 min

Figure 2. Representative traces of absorbance changes that show induction of passive swelling by dinoseb of intact spinach chloroplasts (B), spinach thylakoids (C), and mung bean mitochondria (D) suspended in isotonic KSCN. The model system is presented diagrammatically in A.


5.

MORELAND ET AL.


91

TABLE V Induction of p a s s i v e s w e l l i n g of i n t a c t spinach c h l o r o p l a s t s , spinach t h y l a k o i d s , and mung bean mitochondria by FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s . Osmoticum n

KSCN

A

Compound

KCl

C h l o r o p l a s t s ^ ^ Thylakoids-!?/ Mitochondria^/

_ Mitochondria*'

i (yM) 5 0


110 200 NEC./ 330 160 110 14 71 15

31 230 NE 200 200 110 14 NE 57

9 31 230 45 59 105 34 180 18

30 110 NE 110 220 190 24 89 38

a/ — Concentration (μΜ) r e q u i r e d to induce an i n c r e a s e i n the s w e l l ing r a t e of 0.02 A i n 1 min, r e l a t i v e to the no-herbicide controls. — / Concentration (μΜ) r e q u i r e d to induce an i n c r e a s e i n the s w e l l ing r a t e o f 0.01 A i n 1 min, r e l a t i v e to the no-herbicide controls. SJ NE = no, or minimal, e f f e c t with concentrations up to 400 μΜ.

the e l e c t r o n t r a n s p o r t i n h i b i t o r s (Table V I ) . The c h l o r o p l a s t s used i n these s t u d i e s contained about 250 nmoles stromal K /mg c h l o r o p h y l l . A slow e f f l u x o f K+ was induced by acetone (1%) used as a s o l v e n t f o r the t e s t compounds. Data shown i n Table VI have been c o r r e c t e d f o r the n o - h e r b i c i d e c o n t r o l e f f l u x (40 nmoles K+/mg chlorophyll·min). Diuron d i d not i n c r e a s e the e f f l u x o f K " s i g n i f i c a n t l y above that induced by acetone. I o x y n i l , 3-CIPC, dinoseb, and 2,3-DCIPC induced s i g n i f i c a n t K+ e f f l u x over a s i m i l a r molar c o n c e n t r a t i o n range. P r o p a n i l had the weakest e f f e c t on K+ e f f l u x . The most a c t i v e compounds were 3-CHPC, which i n i t i a t e d e f f l u x a t concentrations below 2 μΜ, and 3,4-DCIPC, which was a s l i g h t l y b e t t e r inducer than FCCP. The r e s u l t s d i r e c t l y support the p o s t u l a t e that the h e r b i c i d e s i n c r e a s e d mem brane p e r m e a b i l i t y to K . +

4

+

The i n h i b i t o r y uncouplers, but not diuron and 2,3-DCIPC, a l s o a l t e r e d the p e r m e a b i l i t y t o protons o f a r t i f i c i a l , p u r e l y l i p o i d a l , liposome membranes. I n the system (Figure 3A), e l e c t r o n s flow



Figure 3. Effects of dinoseb on increasing the rate of reduction of ferricyanide, included within egg yolk phosphatidyl choline liposomes, by ferrocene (B). The model system is presented diagrammatically in A.

5.

MORELAND ET AL.


93

TABLE VI E f f e c t s of FCCP, s e l e c t e d h e r b i c i d e s , and c a r b a n i l a t e s on the e f f l u x of potassium from i n t a c t spinach c h l o r o p l a s t s and the p e r m e a b i l i t y of l e c i t h i n liposomes

K Compound

FCCP Dinoseb Diuron Ioxynil Propanil 3-CIPC 3-CHPC 2.3- DCIPC 3.4- DCIPC

+

e f f l u x from a/ chloroplasts^

22 110 NE£' 110 290 100 6 120 19

H

+

permeability

of liposomes-^

.05 1 NE 3 120 360 49 NE 140

a/ + — Concentration (yM) r e q u i r e d to induce e f f l u x of Κ at a r a t e of 100 nmoles/mg chlorophyll·2 min above the no-herbicide control rate. — ^ Concentration (yM) r e q u i r e d to i n c r e a s e the r a t e of f e r r i cyanide r e d u c t i o n to 80 nmoles/min. The no-herbicide c o n t r o l r a t e was about 40 nmoles/min. c/ — NE = no, or minimal, e f f e c t with concentrations up to 400 yM.

from ascorbate (outside) v i a ferrocene ( i n the liposome membrane) to f e r r i c y a n i d e (included w i t h i n the liposome) i n the presence of tetraphenylboron (18). D i f f u s i o n of H+" across the liposome mem brane i s r e q u i r e d to maintain e l e c t r o n e u t r a l i t y . Dose/response curves were developed from t r a c e s such as shown i n F i g u r e 3B f o r dinoseb. For comparative purposes, the concen t r a t i o n of compound r e q u i r e d to i n c r e a s e the r a t e of f e r r i c y a n i d e r e d u c t i o n to twice that o f the no-herbicide c o n t r o l r a t e are shown i n the l a s t column of Table V I . Uncouplers such as FCCP a c c e l erate the r a t e of f e r r i c y a n i d e r e d u c t i o n , presumably by s h u t t l i n g protons across the membrane i n response to the e l e c t r i c a l poten t i a l generated by the r e d u c t i o n of f e r r i c y a n i d e by ferrocene (28). In t h i s study, FCCP was the most e f f e c t i v e compound. The two phenolic h e r b i c i d e s (dinoseb and i o x y n i l ) were more a c t i v e than p r o p a n i l and chlorpropham. Among the c a r b a n i l a t e s , 3-CHPC and 3,4-DCIPC were more a c t i v e than the parent compound (3-CIPC), whereas 2,3-DCIPC was e s s e n t i a l l y i n a c t i v e . These r e s u l t s



94

demonstrate that the i n h i b i t o r y uncoupler h e r b i c i d e s , l i k e the uncoupler FCCP, can i n c r e a s e the p e r m e a b i l i t y of an a r t i f i c i a l , p u r e l y l i p o i d a l membrane to IT*". In the liposome experiments as reported here, the noh e r b i c i d e c o n t r o l r a t e was a f f e c t e d by the r e l a t i v e concentration of ferrocene and t e t r a p h e n y l boron i n the r e a c t i o n mixture. A d d i t i o n a l l y , responses induced by the compounds v a r i e d q u a n t i t a t i v e l y with the source and p u r i t y of the phosphatidyl c h o l i n e from which the liposomes were prepared. However, the r e l a t i v e order of a c t i v i t y of the compounds remained constant. Data were reported f o r the egg y o l k p r e p a r a t i o n because i t provided a b e t t e r e v a l u a t i o n of the r e l a t i v e a c t i v i t i e s of the c a r b a n i l a t e s . Conclusions From the s t u d i e s and r e s u l t s reported h e r e i n , the f o l l o w i n g general conclusions and e x t r a p o l a t i o n s can be made: a. The s t r u c t u r e / a c t i v i t y c o r r e l a t i o n s apply to c h l o r o p l a s t s , t h y l a k o i d s , mitochondria, and liposomes, i . e . , the responses observed were not membrane s p e c i f i c . b. The i n h i b i t i o n of valinomycin-induced s w e l l i n g ( a t t r i b u t e d to decreased membrane " f l u i d i t y " ) and i n d u c t i o n of passive s w e l l i n g ( a t t r i b u t e d to increased p e r m e a b i l i t y of the membranes to K ) are not o r g a n e l l e s p e c i f i c responses. c. The l i m i t e d s t r u c t u r e / a c t i v i t y s t u d i e s conducted with the c a r b a n i l a t e s suggest that a f f i n i t y f o r b i n d i n g to the Β p r o t e i n complex c o r r e l a t e s with i n c r e a s e d uncoupling a c t i v i t y i n PS I assays, increased uncoupling a c t i v i t y i n mitochondria, enhanced membrane p e r t u r b a t i o n s , enhanced p e r m e a b i l i t y of c h l o r o p l a s t s to K+, and enhanced p e r m e a b i l i t y of liposomes to H+. d. The a c t i o n on photophosphorylation may be separate and independent from b i n d i n g to the Β p r o t e i n complex. e. The i n h i b i t o r y uncouplers may conceivably perturb a l l c e l l u l a r membranes (plasmalemma, t o n o p l a s t , n u c l e a r , and endo plasmic r e t i c u l u m i n a d d i t i o n to the c h i o r o p l a s t and mitochondrial membranes). However, marker systems that can be monitored r e a d i l y which r e f l e c t the p e r t u r b a t i o n s remain to be i d e n t i f i e d . This physiochemical i n t e r a c t i o n of the i n h i b i t o r s with l i p i d s can be expected to a l t e r the many t r a n s p o r t , b i o s y n t h e t i c , and r e g u l a t o r y a c t i v i t i e s a s s o c i a t e d with c e l l u l a r membranes and could be i n volved i n the expression of p h y t o t o x i c i t y . The r e l a t i o n between membrane p e r t u r b a t i o n s and the expres s i o n of p h y t o t o x i c i t y remains to be i d e n t i f i e d . In some of the experiments r e p o r t e d , concentrations i n the 1 0 0 to 2 0 0 yM range were r e q u i r e d to produce I 5 0 responses. However, i n many of these s t u d i e s , i n i t i a l e f f e c t s could be detected at concentrations of 1 to 1 0 yM f o r many of the compounds. At t h i s time, i t i s not p o s s i b l e to determine the impact of minor a l t e r a t i o n s to the p e r m e a b i l i t y and " f l u i d i t y " of o r g a n e l l e membranes on the p h y s i o l o g i c a l s t a t u s of an organism. Conceivably, small changes, +


5.

MORELAND ET AL.


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coupled w i t h a r e d u c t i o n i n the a v a i l a b i l i t y of c h i o r o p l a s t and m i t o c h o n d r i a l l y generated ATP, could have a s i g n i f i c a n t and d r a s t i c e f f e c t over a time span of s e v e r a l hours or days. Acknowledgements This was a cooperative i n v e s t i g a t i o n of the North C a r o l i n a A g r i c u l t u r a l Research S e r v i c e and the United States Department of A g r i c u l t u r e , A g r i c u l t u r a l Research S e r v i c e , R a l e i g h , N . C . Paper No. 7079 of the J o u r n a l S e r i e s of the North C a r o l i n a A g r i c u l t u r a l Research S e r v i c e , R a l e i g h , N . C . The study was supported i n p a r t by P u b l i c H e a l t h S e r v i c e Grant ES 00044. A p p r e c i a t i o n i s ex tended to F . S. Farmer f o r t e c h n i c a l a s s i s t a n c e w i t h the mitochon d r i a l phases of the study.

Literature Cited 1. Moreland, D. E. Annu. Rev. Plant Physiol. 1980, 31, 597-638. 2. Moreland, D. E.; Hilton, J. L. in "Herbicides: Physiology, Biochemistry, Ecology", Vol. 1; Audus, L. J., Ed.; Academic Press: London, 1976; pp. 493-523. 3. Tischer, W.; Strotman, H. Biochim. Biophys. Acta 1977, 460, 113-25. 4. Pfister, K.; Arntzen, C. J . Z. Naturforsch. 1977, 34c, 9961009. 5. Steinback, Κ. E.; Pfister, K.; Arntzen, C. J. Chapter 3 in this book. 6. Shipman, L. L. Chapter 2 in this book. 7. Van Assche, C. J.; Carles, P. M. Chapter 1 in this book. 8. Moreland, D. E.; Huber, S. C. in "Plant Mitochondria"; Ducet, G.; Lance, C., Eds.; Elsevier/North Holland Biomedical Press: Amsterdam, 1978; pp. 191-8. 9. Moreland, D. E.; Huber, S. C. Pestic. Biochem. Physiol. 1979, 11, 247-57. 10. Moreland, D. E. Pestic. Biochem. Physiol. 1981, 15, 21-31. 11. Ducruet, J . M.; Gauvrit, C. Weed Res. 1978, 18, 327-34. 12. Moreland, D. E. in "Progress in Photosynthesis Research", Vol III; Metzner, H., Ed.; Int. Union Biol. Sci.: Tubingen, 1969; pp. 1693-1711. 13. Lilley, P. McC; Walker, D. A. Biochim. Biophys. Acta 1974, 368, 269-78. 14. Armond, P. Α.; Arntzen, C. J.; Briantais, J.-M.; Vernotte, C. Arch. Biochem. Biophys. 1976, 175, 54-63. 15. MacKinney, G. J . Biol. Chem. 1941, 140, 315-22. 16. Lanzetta, P. Α.; Alvarez, L. J.; Reinach, P. S.; Candia, O.A. Anal. Biochem. 1979, 100, 95-7. 17. Chance, B.; Williams, G. R. J . Biol. Chem. 1955, 217, 409-27. 18. Hinkle, P. Biochem. Biophys. Res. Commun. 1970, 41, 1375-81. 19. Alsop, W. R.; Moreland, D. E. Pestic. Biochem. Physiol. 1975, 5, 163-70.


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20. Good, Ν. E. in "Encyclopedia of Plant Physiology, New Series", Vol. 5; Trebst, Α.; Avron, Μ., Eds.; Springer-Verlag: Berlin, 1977; pp. 429-36. 21. Fiolet, J. T. W.; Bakker, E. P.; Van Dam, K. Biochim. Biophys. Acta 1974, 368, 432-45. 22. Moreland, D. E.; Huber, S. C.; Novitzky, W. P. Proc. 5th Int. Congr. Photosynth.: Halkidiki, Greece, 1981; in press. 23. Chappel, J . B.; Crofts, A. R. in "Regulation of Metabolic Processes in Mitochondria"; Tager, J. M.; Pappa, S.; Quagliariello, E.; Slater, E. C., Eds.; Elsevier: Amsterdam, 1966; pp. 293-316. 24. Jagendorf, A. T. in "Bioenergetics of Photosynthesis"; Govindjee, Ed.; Academic Press: New York, 1975; pp. 413-92. 25. Pressman, B. C. Annu. Rev. Biochem. 1976, 45, 501-29. 26. Huber, S. C.; Moreland, D. E. Plant Physiol. 1979, 64, 115-9. 27. Cavalieri, A. J.; Huang, A. H. C. Plant Physiol. 1980, 66, 588-91. 28. Bakker, E. P.; Van Den Heuvel, E. J.; Wiechmann, A. H. C.; Van Dam, K. Biochim. Biophys. Acta 1973, 292, 78-87. RECEIVED

September 21, 1981.


Interaction of Herbicides with Cellular and Liposome Membranes

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