Conformation and Activity of Chloroplast Coupling ... - life.illinois.edu
328
Biochimica et Biophysica Acta, 5 4 8 ( 1 9 7 9 ) 3 2 8 - - 3 4 0 © Elsevier/North-Holland
Biomedical Press
BBA 47747
CONFORMATION AND ACTIVITY OF CHLOROPLAST COUPLING FACTOR EXPOSED TO LOW CHEMICAL POTENTIAL OF WATER IN CELLS
H A S S A N M. Y O U N I S *, J O H N S. B O Y E R * * a n d G O V I N D J E E
Departments of Botany, Agronomy, and Physiology and Biophysics and United States Department of Agriculture, Science and Education Administration, Agricultural Research, 289 Morrill Hall, University of Illinois, Urbana, IL 61801 (U.S.A.) (Received November 10th, 1978) (Revised manuscript received April 10th, 1979}
Key words: Photophosphory lation; Coupling factor; Chemical potential; A TPase ; Conformation; Nucleotide binding
Summary (1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (~w) of--25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low xPw had less Ca2+-ATPase activity than control CFI and did not recouple phosphorylation in CF1deficient chloroplasts. In contrast, CF~ from leaves having high ~w only partially recoupled phosphorylation by CFrdeficient chloroplasts from leaves having low ~w. This indicated that low ~w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low ~w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that * Present address: D e p a r t m e n t of Plant Protection, Faculty of Agriculture, and Biochemistry and Molecular Biology, University Science Center, Univeristy of Alexandria, Egypt. ** Address reprint requests to J.S. Boyer, USDA/SEA/AR, D e p a r t m e n t of Botany, 289 Morrll Hall, University of Illinois, Urbana, IL 6 1 8 0 1 , U.S.A. Abbreviations: ~ w , water potential CF l , coupling factor protein (ATP synthetase when attached to the membrane); e-ADP, 1,N6-ethenoadenosine diphosphate; e-ATP, 1,N6-etlienoadenosine triphosphate; EDTA, ethylenediaminetetraacetic acid; Tricine, N-tris(hydroxymethyl)methylglycine.
329 CFI from leaves having low ~w differed from control CF~. The CF~ from leaves having low ~w also had decreased ability to bind fluorescent nucleotides (e-ATP and e-ADP). (4) Exposure of isolated CF~ to low ~/w in vitro by preincubation in sucrosecontaining media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2÷ in the preincubation medium markedly inhibited Ca2÷-ATPase activity. (5) These results show that CF~ undergoes changes in cells which alter its phosphorylating ability. Since low cell ~/w changed the spectroscopic properties but not other protein properties of CFI, the changes were most likely caused by altered conformation of the protein. This decreased the binding of nucleotides and, in turn, photophosphorylation. The inhibition of ATPase activity in CF~ in vitro at low ~w and high ion concentration mimicked the change in activity seen in vivo.
Introduction
The chemical potential of water (hereafter described in terms of the water potential, ~w) * decreases when cell water content decreases [1]. In the chloroplast-containing cells of plants, the decrease is associated with large losses in chloroplast activities, notably electron transport and photophosphorylation [2--5]. The losses occur at ~w that are commonly encountered under natural conditions [2,3] and may account for some of the decrease in rates of photosynthesis observed at low ~w [2,5]. Fellows and Boyer [4] presented evidence that the inhibition was correlated with changes in the thickness of the lamellar membranes of the thylakoids in energized chloroplasts. The changes in thickness could be seen in vivo and persisted in vitro. However, the nature of the membrane structure that accounts for the changes in thickness has not been identified. In this paper, we present evidence that the molecular basis of these changes is attributable in part to changes in the conformation of chloroplast coupling factor (CF1). Structural transformations of CF~ have been demonstrated by the ability of the protein to trap tritiated water [6,7] and to bind N-ethylmaleimide [ 8,9], adenine nucleotides [ 10--12], and trinitrobenzenesulfonate [13] when the thylakoid membranes are energized in vitro. With the demonstration that CFl-bound nucleotides undergo a light-dependent and uncouplersensitive exchange with free nucleotides [11,14], it has been proposed that the structural transformations are conformational changes in CF1 that induce changes in the binding affinities for ADP, Pi, and ATP during photophosphorylation [15,16]. Results in the present work indicate that changes in CFI conformation also occur within cells, persist after isolation, and lead to altered
* T h e w a t e r p o t e n t i a l is r e l a t e d t o t h e c h e m i c a l p o t e n t i a l 0~, e r g s • m o 1 - 1 ) o f w a t e r a c c o r d i n g t o xP w ffi (~uw - - / ~ 0 ) / V , w h e r e t h e subscripts w a n d 0 r e f e r t o t h e s y s t e m and the r e f e r e n c e , r e s p e c t i v e l y , a n d V is t h e p a r t i a l m o l a l v o l u m e o f l i q u i d w a t e r ( c m 3 • t o o l -1 , v i r t u a l l y a c o n s t a n t o v e r t h e range o f xIJw i n b i o l o g i c a l s y s t e m s ) . F o r c o n v e n i e n c e , w e r e p o r t ~ w i n p r e s s t t r e u n i t s , 1 b a r ffi 1 0 6 e r g s • c m - 3 = 0 . 9 8 7
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330 affinities for nucleotides that may account for alterations in phosphorylating activity. Materials and Methods
Plant material and ~w. Commercial spinach (Spinacea oleracea L.) was purchased from a local market. Different leaf ~w were obtained by partial drying of the leaves for 1-.--3 h in the light in a controlled environment room (temperature = 29°C; relative humidity = 40--60%; irradiance = 150 W" m -~ from fluorescent (daylight) bulbs). Leaf qJw was measured with a thermocouple psychrometer by the isopiestic technique described by Boyer and Knipling [17]. Measurement o f chloroplast activities. Chloroplasts were isolated from the leaves as described by Lien and Racker [18]. Chloroplast Ca2+-ATPase was activated by heat at 6 0 6 4 ° C for 4 min in the presence of ATP (20 mM) and dithiothreitol (5 mM), and chloroplast Mg2+-ATPase was activated by incubation with dithiothreitol (25 mM) for 15 min. Activities of ATPase were measured as described by Lien and Racker [18] for Ca2+-dependent ATPase, and by replacing Ca 2÷ with 5 mM MgC12 for Mg2+-dependent ATPase. Phenazine methosulphate-mediated cyclic photophosphorylation was measured potentiometrically with a recording pH meter as described by Dilley [19]. Illumination was with saturating (180 W • m -2 between 400 and 700 nm) red light (maximum irradiance at 668 nm), passed through a heat filter [5]. Coupling factor was prepared by extraction with chloroform according to Younis et al. [20]. The protein was stored at 4°C in 2 M (NH4)2SO4, 2 mM ATP, 1 mM EDTA, and 20 mM Tricine/OH-, pH 8, until needed for measurements. Prior to use, the suspension was centrifuged (14 000 × g), resuspended in 40 mM Tricine/OH-, pH 8, recentrifuged (14 000 ×g), and desalted on a Sephadex G-50 column (20 × 1 cm). This procedure provided a protein of high specific activity relatively free of other proteins (Ref. 20, and see Fig. 3 below). The Ca2÷-ATPase of the protein was heat activated at 6 0 - 6 4 ° C for 4 min and assayed as for the chloroplasts. Chlorophyll was determined spectrophotometrically in 80% acetone extracts [21]. Protein concentration was measured usually by the m e t h o d of Lowry et al. [22] and sometimes spectrophotometrically [23]. In the former method, the concentration was multiplied by 1.15 and, in the latter, by a factor of 1.85 to express the protein concentration on a dry weight protein basis [24]. For preparation of CFl-depleted chloroplasts, the chloroplasts were treated with EDTA as described by McCarty [25]. Phosphorylation was reconstituted in these chloroplasts by adding CF1, and phenazine methosulfate-mediated cyclic photophosphorylation was assayed as described in Table II. Polyacrylamide gel electrophoresis of the protein was performed as described by Weber and Osborn [26]. Fluorescence measurements. Fluorescence polarization of CF1 was measured with a photon-counting polarization p h o t o m e t e r in the laboratory of G. Weber. This instrument (see Weber and Bablouzian [27] and Jameson et al. [28]) allows simultaneous monitoring of the parallel and perpendicular components of the polarized fluorescence by using two photomultipliers at right angles to
331 the exciting beam. It also permits convenient subtraction of background scattering at low signal-to-noise values. The excitation wavelength was 280 nm and the emitted fluorescence was filtered through Coming * glass C.S. 0-54 filters. The binding of fluorescent nucleotides (e-ADP and e-ATP) to CFI was followed b y measuring the polarization of the average free and b o u n d e-nucleottide fluorescence as a function of e-nucleotide concentration [29]. The excitation wavelength was 310 nm (passed through a Coming glass C.S. 7--54 filter) and the emission at 405 nm (filtered through a Coming glass C.S. 3-75 filter) was measured. Circular dichroism measurements. The circular dichroism spectrum of CF~ in 20 mM Tricine/OH-, pH 8.0, was measured between 200 and 250 nm with a JASCO model J 4 0 A Automatic Recording Spectropolarimeter. The spectropolarimeter was calibrated with D-(+)-camphorsulfonic acid; the pathlength was 1 mm; the protein concentration was 0.5 mg • m1-1, and the temperature was 25°C. The mean residue molecular weight was taken to be 114. Ultraviolet spectra. Ultraviolet absorption spectra of the protein were measured in an Aminco s p e c t r o p h o t o m e t e r DW-2. Results The photophosphorylating activity of spinach chloroplasts decreased with decreasing leaf q]w (Fig. 1). In chloroplasts from leaves with a q z of a b o u t - 2 5 bars, the rate of cyclic photophosphorylation was 45% of that in the control chloroplasts. A similar result was found with sunflower leaves, in which photophosphorylation was markedly inhibited and uncoupled from electron flow at low ~w [3]. To determine whether the effect involved the phosphorylating enzyme CF1 or other aspects of phosphorylation, we assayed for Ca2÷-depen dent and Mg2*-dependent ATPase activity of the chloroplasts. There was a linear decrease in the Ca2*-ATPase activity of the chloroplasts as leaf q]w decreased (47% of control at ~ w of --25 bars, Fig. 2, solid circles). The Mg 2÷dependent ATPase activity also decreased with decreasing ~w (Fig. 2, open squares). The Ca2÷-ATPase activity recovered fully if the leaves were rehydrated before the chloroplasts were isolated (Fig. 2, open circle). The above results suggest that some of the effects of low q]w on photophosphorylation were caused by an effect on CF~ itself. Therefore, we studied CF~ from leaves with various ~ . Table I shows that CF1 isolated from chloroplasts of leaves having t w o different ~w yielded the same a m o u n t of CFI protein b u t had different Ca2÷-ATPase activities. Activity of the protein from leaves with low ~w was much lower (23% of control) than for protein from leaves with high ~w when the ATPase was n o t heat activated (Table I). However, the relative difference in activity was reduced after heat activation (Table I). Since the protein was probably closest to the in vivo form before heat activation, we
o f a t r a d e m a r k or p r o p r i e t a r y p r o d u c t d o e s n o t c o n s t i t u t e a g u a r a n t e e o r w a r r a n t y o f t h e p r o d u c t b y t h e U . S . D e p a r t m e n t o f A g r i c u l t u r e and d o e s n o t i m p l y its approval t o t h e e x c l u s i o n o f o t h e r p r o d u c t s that m a y a l s o b e s u i t a b l e .
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F i g . 1. P h o t o p h o s p h o r y l a t i n g a c t i v i t y o f c h l o r o p l a s t s i s o l a t e d f r o m s p i n a c h (S. oleracea L.) leaves w i t h various w a t e r potentials. Control chloroplasts were isolated f r o m leaves of the s a m e p o p u l a t i o n w i t h o u t d e s i c c a t i o n . T h e c o n t r o l r a t e w a s 1 0 6 0 # t o o l A T P " h -1 • m g - l Chl, p h e n a z i n e m e t h o s u l f a t e c o n c e n t r a t i o n w a s 15 # M a n d c h l o r o p h y l l c o n c e n t r a t i o n w a s 20 # g • m1-1 . F i g . 2. T h e C a 2 + - A T P a s e ( e ) a n d M g 2 + - A T P a s e (~) a c t i v i t i e s o f c h l o r o p l a s t s i s o l a t e d f r o m s p i n a c h l e a v e s with various water potentials. Mg2+-ATPase was activated with dithiothreitol. Recovery from low water P o t e n t i a l (o) s h o w s t h e a c t i v i t y o f C a 2 + - A T P a s e a f t e r r e h y d r a t i o n o f t h e t i s s u e f r o m a w a t e r p o t e n t i a l o f --24.5 bars.
used CF1 t h a t had n o t been heat activated during the following measurements, except where stated.
Coupling activity of the isolated proteins Table II summarizes the results of an experiment in which chloroplasts from leaves with high and low ~w were depleted of CF~ by EDTA treatment, then
TABLE I Y I E L D A N D Ca2+-ATPase A C T I V I T Y O F C O U P L I N G F AC T OR ISOL AT E D FR OM C HL OR OPL AST S OF LEAVES WITH HIGH (CONTROL) AND LOW WATER POTENTIALS E x p e r i m e n t a l c o n d i t i o n s as i n M a t e r i a l a n d M e t h o d s . Treatment
Control Water deficient
Leaf water potential (bars)
---3 --17
Total chlorophyll (mg • extract -I )
15.8 15.2
Total protein (rag extract -I )
8.8 8.9
Protein released/ chlorophyll ( m g • m g -1 )
0.55 0.58
Ca2 +-ATPase a c t i v i t y ~umol Pi " m i n - I " m g - I protein) Before heat activation
After heat activation
2 0.46
14 11
333 T A B L E II R E C O N S T I T U T I O N OF P H O T O P H O S P H O R Y L A T I O N IN C O U P L I N G F A C T O R - D E F I C I E N T CHLOR O P L A S T S BY C O U P L I N G F A C T O R F R O M L E A V E S W I T H H I G H ( C O N T R O L ) A N D L O W W A T E R POTENTIALS P h o s p h o r y l a t i o n was r e c o n s t i t u t e d b y c o m b i n i n g t h e E D T A - t r e a t e d c h l o r o p l a s t s w i t h 1 m l o f t h e i r s u p e r n a t a n t ( c o n t a i n i n g CF l ) to give a c h l o r o p h y l l c o n c e n t r a t i o n o f 0 . 2 r n g / m l . MgCl 2 ( 1 0 m M ) was a d d e d a f t e r the E D T A - t r e a t e d c h l o r o p l a s t s h a d b e e n a d d e d a n d t h e m i x t u r e w a s i n c u b a t e d f o r 15 rain b e f o r e assay at r o o m t e m p e r a t u r e . A t t h e s a m e t i m e , a l i q u o t s o f t h e u n t r e a t e d c h l o r o p l a s t s were d i l u t e d to t h e c h l o r o p h y l l c o n c e n t r a t i o n of t h e t r e a t e d s a m p l e s . P h o s p h o r y l a t i n g a c t i v i t i e s w e r e meastLred w i t h 10 p g c h l o r o p h y l l / m l in a 2 m l r e a c t i o n m i x t u r e c o n t a i n i n g : 3 m M N a H 2 P O 4, 3 m M MgC12, 17 m M KC1, 1.5 m M A D P a n d 5 0 pM p h e n a z i n e m e t h o s u l f a t e , p H 7.8. P r o t e i n c o n c e n t r a t i o n in b o t h s u p e r n a t a n t s was 0 . 1 4 m g / m l as d e t e r m i n e d b y t h e m e t h o d o f L o w r y et al. [ 2 2 ] . Treatment
1. U n t r e a t e d c h l o r o p l a s t s f r o m leaves w i t h a ~Isw o f a. --1 b a r ( c o n t r o l s ) b. - - 1 8 b a r s 2. E D T A - t r e a t e d c h l o r o p l a s t s f r o m leaves w i t h a ~ w o f a. --1 b a r ( c o n t r o l s ) b. - - 1 8 b a r s 3. E D T A - t r e a t e d c h l o r o p l a s t s f r o m l e a v e s w i t h a ~ w o f --1 b a r ( c o n t r o l s ) plus a. CF 1 f r o m leaves w i t h ~ w o f --1 b a r b . CF 1 f r o m leaves w i t h ~ w o f - - 1 8 b a r s 4. E D T A - t r e a t e d c h l o r o p l a s t s f r o m l e a v e s w i t h ~ w o f - - 1 8 b a r s plus a. CF 1 f r o m leaves w i t h ~ w o f --1 b a r b. CF 1 f r o m leaves w i t h ~ w o f - - 1 8 b a r s
Cyclic p h o t o phosphorylation (pmol ATP • h -1 • m a -1 Chl)
1060 440 460 160 700 400 220 170
reconstituted by adding back the CFl-containing supernatant. The CFI released from leaves with low ~w did not restore phosphorylation in CFl-depleted chloroplasts from control leaves (Table II, cf. lines 2a, 3a and b). This result indicates a specific effect of low ~w on CF1. However, control CFI restored phosphorylation only slightly in CF~-depleted chloroplasts from leaves with low ~w (Table II, cf. lines 2b, 4a and 3a). Therefore, in addition, to a specific effect on CF~, low ~w also altered the membrane.
Electrophoretic mobility and subunit structure Coupling factor placed on polyacrylamide gels exhibited a single band with a mobility unaltered by ~w (RF of 0.43 for CFI from leaves with ~w o f - 2 and --18 bars). When detergent was added to the gels to separate the protein subunits, five subunits were always present (Fig. 3), each with an R F unaffected by ~w.
Polarization of CF1 fluorescence The lifetime of the excited state o f fluorescence in molecules is o n the order of 10 -8 s [ 3 0 , 3 1 ] . Because the relaxation times for the rotation o f fluorescent macromolecules are of the same order o f magnitude, their emitted fluorescence is partially polarized, and the degree o f polarization affords a convenient means of determining their relaxation time under a variety o f circumstances [ 3 0 , 3 2 ] . In identical media, and with the assumption that the lifetime o f the excited
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Fig. 3. Densitometrie scans along polyacrylamide gels showing the subunits of (a) CFI isolated from s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f ---2 bars a n d (B) CF 1 i s o l a t e d f r o m s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f - - 1 8 b a r s . T h e p r o t e i n s u b u n i t s (~ - - e) w e r e s e p a r a t e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in t h e p r e s e n c e o f s o d i u m d o d e c y l sulfate, Gels w e r e l o a d e d w i t h 50 #g o f p r o t e i n ( A ) or 70 # g o f p r o t e i n (B) in 4 0 m M T r i e i n e / O H - , p H 8, b u f f e r . Fig. 4. Circular d i c h r o i s m s p e c t r a o f c o u p l i n g f a c t o r i s o l a t e d f r o m s p i n a c h leaves w i t h w a t e r p o t e n t i a l s of - - 2 . 9 b a t s or - - 1 8 . 7 bars. T h e p a t h l e n g t h was 1 t a r a , the p r o t e i n c o n c e n t r a t i o n w a s 0 . 5 rag • m l - ! • a n d t h e b u f f e r was 20 m M T r i c i n e / O H - , p H 8.0.
state is constant, differences in polarization of a particular substance are caused by differences in conformation. Table III shows that the degree of polarization of fluorescence in CFI from leaves with low ~w (--16 bars) was less by about 0.036 (+0.003) than in CFI from the control leaves (--1 bar). Circular dichroism spectra In the ultraviolet region between 200 and 250 nm, circular dichroism spectra are uniquely determined by protein conformation. Although a number of properties of the protein contribute to the spectra in this region, the quantity
TABLE III FLUORESCENCE POLARIZATION OF COUPLING FACTOR PROTEIN ISOLATED FROM SPINACH LEAVES WITH DIFFERENT WATER POTENTIALS T h e p r o t e i n was d e s a l t e d o n a S e p h a d e x G - 5 0 ( 2 0 × 1 c m ) c o l u m n . T h e b u f f e r was 4 0 m M t r i c i n e / O H , p H 8. P r o t e i n in the s a m e b u f f e r at a c o n c e n t r a t i o n o f 0 . 4 2 m g / m l was u s e d f o r m e a s u r i n g t h e p o l a r i z a t i o n o f f l u o r e s c e n c e as d e s c r i b e d in Materials a n d M e t h o d s . D e g r e e of p o l a r i z a t i o n is F I I - F.L/FI[ + F±, w h e r e FI[ a n d FJ. are t h e f l u o r e s c e n c e i n t e n s i t y parallel a n d p e r p e n d i c u l a r , r e s p e c t i v e l y , t o t h e e x c i t i n g r a d i a t i o n . I n s t r u m e n t f l u c t u a t i o n s i n t r o d u c e e r r o r of ~ 0 . 0 0 3 in p o l a r i z a t i o n values. Water potential of leaves (bars) --1
--16
Degree of polarization of fluorescence by isolated coupling factor
0.306 0.270
335
of s-helix, the orientation of secondary (~) structure, and the arrangement of 'randomly' coiled regions of the protein are particularly contributory. For CF1 from leaves having ~w of --2.9 and --18.7 bars, the molecular ellipticity (0) differed in the same buffer and at the same protein concentration (Fig. 4). Both proteins exhibited a similar extremum around 208 nm, which indicates that both contained a similar amount of s-helix (21% as calculated from Greenfield and Fasman [33]). However, CFI from leaves with ~w of--18.7 bars had a smaller molecular ellipticity between 208 and 230 nm than CF, from control leaves, which suggests that ~ structure and randomly coiled regions had been altered by treatment at low ~w [33].
Ultraviolet absorption spectra The CF, isolated from leaves with ~w of --17 bars absorbed less in the ultraviolet region of the spectrum than did CF1 from leaves with high *w (Fig. 5). The differences in the spectra peaked at 230 and 271 nm {Fig. 5, inset). Because these differences occurred in proteins having the same number of subunits each having essentially the same mobility on polyacrylamide gels, the amino acid composition of CF, was unlikely to have been substantially altered by ~w treatment. Therefore, the differences in spectra were probably caused
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210 230 250 270 290 310 .330 350 Wovelength (nm) Fig. 5. U l t r a v i o l e t a b s o r p t i o n s p e c t r a o f c o u p l i n g f a c t o r f r o m s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f ( A ) - - 2 bars a n d (B) - - 1 7 bars. Inset: T h e s p e c t r a l d i f f e r e n c e b e t w e e n ( A ) and (B). T h e l o w e s t trace in the m a i n figure is f o r t h e b u f f e r . T h e p r o t e i n c o n c e n t r a t i o n w a s 1.2 r n g / m l in 4 0 m M T r i c i n e / O H - , p H 8.
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14
16
Fig. 6. P o l a r i z a t i o n o f e - A T P a n d e - A D P f l u o r e s c e n c e b y c o u p l i n g f a c t o r a t v a r i o u s e - A T P a n d e - A D P c o n c e n t r a t i o n s . ( A ) e - A T P plus c o u p l i n g f a c t o r p r o t e i n f r o m c o n t r o l leaves w i t h w a t e r P o t e n t i a l of --2 bars ( e ) o r - - 2 5 b a r s (o). Inset: e - A T P plus c o u p l i n g f a c t o r (a s e p a r a t e p r e p a r a t i o n ) f r o m c o n t r o l leaves w i t h w a t e r p o t e n t i a l o f --2 bars (m) o r - - 1 7 bars ( a ) . (B) e - A D P plus c o u p l i n g f a c t o r f r o m leaves w i t h w a t e r p o t e n t i a l o f --2 bars ( c o n t r o l ) ( e ) o r - - 2 5 bars (o). T h e m e d i u m c o n t a i n e d 1.4 # M p r o t e i n a n d 40 m M T r i c i n e / O H - , p H 8.
TABLE IV E F F E C T O F W A T E R P O T E N T I A L A N D Mg 2+ ON H E A T - A C T I V A T E D Ca2÷-ATPase A C T I V I T Y O F COUPLING FACTOR T h e p r o t e i n f r o m l e a v e s w i t h a high ~ w w a s i n c u b a t e d for 30 m i n b e f o r e t h e a s s a y at r o o m t e m p e r a t u r e ( A ) b e f o r e h e a t a c t i v a t i o n or (B) a f t e r h e a t a c t i v a t i o n . I n p r e p a r a t i o n f o r i n c u b a t i o n , a s a m p l e o f a m m o n i u m sulfate s u s p e n s i o n o f CF 1 was c e n t r i f u g e d a n d t h e p e l l e t was dissolved in 4 0 m M T r i c i n e / O H - . p H 8, a n d r e c e n t r i f u g e d a g a i n a t 14 0 0 0 X g f o r 10 re.in. T h e c l e a r s u p e r n a t a n t was d e s a l t e d o n a 20 × 1 c m Sep h a d e x G - 5 0 c o l u m n . S u c r o s e was a d d e d as solid sucrose a n d MgC12 was a d d e d f r o m a 1 M s t o c k s o l u t i o n . A f t e r t h e i n c u b a t i o n , Ca2+-ATPase a c t i v i t y was d e t e r m i n e d a f t e r a d d i t i o n of 20 #l o f t h e i n c u b a t i o n m i x t u r e t o 1 m l o f t h e assay m e d i u m . T r e a t m e n t ( c o m p o n e n t added to p r o t e i n in 4 0 m M T r i c i n e / O H - , p H 8)
None 0.8 M 5 mM 0.8 M 10 mM
sucrose MgCI 2 s u c r o s e + 5 m M MgC12 MgCl 2
0.8 M suCrose + 10 mM MgCI 2
Ca2÷-ATPase a c t i v i t y ( # m o l Pi " m g -1 p r o t e i n • rain - 1 ) (A) T r e a t m e n t b e f o r e heat activation
(B) T r e a t m e n t a f t e r heat activation activation
20 28 21 13 13
14 7 13
--
5 3
337 by a difference in orientation of ultraviolet-absorbing groups on, or light scattering by, the proteins. Binding o f e-ADP and e-ATP Both e-ADP and e-ATP are fluorescent and bind to CF, [29,34]. They can substitute to a considerable degree for ADP and ATP in many reactions in chloroplasts and isolated CF, [29,34--36]. We tested the ability of CF, from leaves with low ~I'w to bind e-ATP and e-ADP by the technique of fluorescence polarization. The fluorescence of these analogs in aqueous solutions, as with many small fluorescent molecules, is n o t polarized because their rotational relaxation times are considerably shorter than their excited lifetimes (approx. 20 ns). The relaxation time is increased by placing the fluorescent molecule in a viscous medium or by attaching the molecule to a macromolecule such as a protein [30,32,37]. As a result, the fluorescence of the adsorbed molecule is partially polarized. Therefore, in any mixture of small fluorescent molecules with a protein, the fraction of the total fluorescence that is polarized will be proportional to the fraction of fluorescent molecules b o u n d to the protein. Under otherwise constant conditions, differences in the binding properties of proteins saturated with the fluorescent molecule represent differences in relaxation times attributable to differences in protein conformation. In the case of e-ADP and e-ATP, differences in binding properties of CF, should reflect differences around the active or regulatory sites for photophosphorylation. Fig. 6 shows the saturation binding of e-ATP and e-ADP to CF, from leaves having various ~I'w in the same buffer and at the same protein concentration. Unlike CF, from leaves with a ~I,~ of --2 bars, which b o u n d large amounts of e-ATP (Fig. 6A), CF, from leaves with a xI,~ of --17 bars b o u n d less e-ATP (Fig. 6A, inset). The dramatic decrease in fluorescence polarization could n o t be attributed to fluorescence quenching by CF,, since the total fluorescence of the control and treated CF, was the same. At xI'w of --25 bars, CF, appeared unable to bind significant e-ATP or e-ADP (Fig. 6). Effects o f Mg 2+ and low ~I'w in vitro on A TPase activity o f isolated CF, During water loss from cells, the concentration of ions around CF, should increase (at a xI'w of --25 bars, the water content of the tissue had decreased to a b o u t 1/3 that in the controls). To test whether prior incubation of CF, in high concentrations of ions could alter the subsequent biological activity of the protein, we preincubated CF, in 5 mM or 10 mM Mg 2+. Preincubation in 5 mM Mg 2+ inhibited the subsequent Ca2+-ATPase activity of CF, only if the CF, had been heat activated (Table IV). At 10 mM, Ca2+-ATPase activity was inhibited regardless of the timing of preincubation in relation to heat activation (Table IV). The direct effect on CF, Of a decrease in the chemical potential o f water was tested b y preincubating CF, in 0.8 M sucrose (~w = - - 2 5 bars). Ca2+-ATPase activity was enhanced if preincubation occurred before heat activation of CF, b u t it was inhibited b y preincubation after heat activation (Table IV). The incubation of CF, in 10 mM Mg 2+ plus 0.8 M sucrose was particularly inhibitory to the Ca2+-ATPase activity of the protein (Table IV). Because the
338 assays measured Ca 2÷ ATPase activity after the incubation, neither sucrose nor Mg2÷ was present in significant quantity during assay. Discussion
These results show that the inhibition of photophosphorylation and ATPase activities of chloroplasts isolated from leaves with low q~w is associated with a change in the spectroscopic properties of coupling factor. Evidence for such a change includes differences in circular dichroism (Fig. 4), polarization of protein fluorescence (Table III), ultraviolet absorption (Fig. 5), and ability to bind nucleotides (Fig. 6). However, there were no significant differences in the yield of protein extractable from the chloroplasts (Table I) or in the number of subunits or the mobility of the protein and its subunits on polyacrylamide gels (Fig. 3). Therefore, the primary structure and molecular weight of the protein were unlikely to have been altered by low ~w, although we cannot exclude the possibility of changes in CF1 binding of other molecules too small to be detected by our gel techniques. With this possible exception, we therefore attribute the spectroscopic differences to changes in the conformation of CF~. Evidence for conformational changes in CF~ has been presented previously for thylakoids energized either by light [6,7], acid-base transitions in the dark [7,13,38], or external voltage pulses [39]. These findings imply that such energy-dependent alterations are required in some way for photophosphorylation. In the present work, CF, was exposed in vivo to low ~w, and the effects on photophosphorylation and conformation persisted after isolation of the protein. Apparently, the conformation of CFI changed in the cells, and the altered conformation was quite stable. Because the effects of photophosphorylation were reversed by rehydration of the cells before isolation of CF~, the altered conformation, although stable, must have been inherently reversible under appropriate conditions in the cells. A similar phenomenon has been seen in chloroplast electron transport; the effect of low ko~ was reversed only if cells were rehydrated before isolation of the plastids [ 5,40,41 ]. The recoupling experiments (Table II) provided a critical confirmation of the effects of low ~Pw on CF,. In these experiments, the protein was unable to restore cyclic photophosphorylation under conditions that readily supported restoration by control CF~. T h e failure of CF~ from low q]w to restore phosphorylation must have been caused by altered biological activity, decreased binding affinity for chloroplast membranes, or both. Clearly, the effect persisted after removal of the protein from the membranes, as had been indicated by the spectroscopic measurements. It is important that control CFI was only slightly able to recouple CF~deficient chloroplasts from leaves with low ~w. Therefore, although low ~w caused a specific effect on CF~, it also altered the chloroplast membranes. Thus, low qJw had multiple effects on chloroplasts, as has been suggested previously by alterations in electron transport and non-cyclic photophosphorylation in chloroplasts from leaves having low ~w [3]. The results (Table II) also add more evidence to what has been discussed by others [42,43] that CF~ added to CF~-deficient chloroplasts has coupling activity in addition to its structural role on the membrane.
339 The most obvious and significant change in CF~ from leaves with a low ~w was the decrease in binding of e-ADP and e-ATP (Fig. 6). It is generally accepted that ATP synthesis involves the binding of ADP and Pi to CF~ and the release of ATP into the medium. Consequently, the marked losses in binding could have contributed to losses in phosphorylating activity at low ~w (Fig. 1). Since concomitant changes in protein conformation also occurred at low q~w, decreased nucleotide binding may have been caused by physical changes in or around the nucleotide binding sites. It should be noted, however, that residual phosphorylation and ATPase activity remained at ~w that markedly reduced binding of fluorescent nucleotides (cf. Figs. 1 and 2 with Fig. 6). The analogs probably bind with less affinity than the normal substrates because of the altered structure of the analogs. In support of this idea, Shahak et al. [35] showed that CFI phosphorylates e-ADP vigorously but the Michaelis constant is about twice that for ADP. The persistence of changes in CF1 after its isolation from cells implies that any attempt to reproduce the effect in vitro must involve a pretreatment of CF~ but assay under uniform conditions to detect effects of the pretreatment. Lowering the water potential of cells increases the concentration of ions in the cytoplasm but also decreases the activity of water molecules. Both effects could bring about conformational changes in CF~. The losses in activity of CF~ after preincubation in high Mg2+ and/or low water activity (Table IV) suggest that CF~ was altered to a different reactive state (Ref. 44 and Younis, H.M., Boyer, J.S. and Govindjee, unpublished results). This new state was stable enough to be subsequently detected by a change in ATPase activity even though the assay was conducted with high water activity and negligible Mg2+. Because the inhibition of ATPase activity in vitro was similar to that in vivo, the in vitro pretreatment seemed to mimic the events occurring within the cells. This conclusion is generally consistent with the behavior of chloroplasts in cells having low ~w. Photosynthetic electron transport and phosphorylation begin to be inhibited 5----10 min [45] after desiccation begins, and recovery after water is resupplied is almost as rapid (15 min [41]). Chloroplast membranes change thickness at the same time but appear normal in other aspects [4]. The rapidity of the changes makes it unlikely that membrane degradation or synthesis is involved and suggest rapid changes in conformation, interaction of membrane subunits, or both. For CF1, the simulation of cell dessication involved rapid changes in water activities and Mg2÷ concentrations over a range likely to occur in cells. The similarity of the simulation and cell dessication results suggest that at least part of the desiccation-induced decrease in the phosphorylating activity of chloroplasts is caused by a simultaneous increase in ion concentration and decrease in chemical potential of water that change the conformation of CF~ and, in turn, decrease the binding of nucleotide substrates to the coupling site(s).
Acknowledgments We are particularly indebted to Dr. Gregorio Weber for help with the fluorescence experiments, the use of his polarization photometer, and for discussions
340
and a critical reading of the manuscript. We also thank Dr. C.J. Arntzen and J. Mullet for assistance in the measurements of ultraviolet absorption spectra. We gratefully acknowledge financial support from the National Science Foundation (J.S.B., PCM 76-11026; G., PCM 76-11657) and the U.S. Public Health Service (G. Weber, GM 11223). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Boyer, J.S. (1969) Annu. Rev. Plant Physiol. 20, 351--364 Boyer, J.S. and Bowen, B.L. (1970) Plant Physiol. 4 5 , 6 1 2 - - 6 1 5 Keck, R.W. and Boyer, J.S. (1974) Plant Physiol. 53, 474--479 Fellows, R.J. and Boyer, J.8. (1976) Planta 132, 229--239 Mohanty, P. and Boyer, J.S. (1976) Plant Physiol. 57, 704--709 Ryrie, I.J. and Jagendorf, A.T. (1971) J. Biol. Chem. 246, 3 7 7 1 - - 3 7 7 4 Ryrie, I.J. and Jagendorf, A.T. (1972) J. Biol. Chem. 247, 4 4 5 3 - - 4 4 5 9 McCarty, R.E., Pittman, P.R. and Tsuehiya, Y. (1972) J. Biol. Chem. 247, 3048--3051 McCarty, R.E. and Fagan, J. (1973) Biochemistry 12, 1503--1507 Roy, H. and Moudrianakis, E.N. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2720--2724 Harris, D.A. and Slater, E.C. (1975) Biochim. Biophys. Aeta 3 8 7 , 3 3 5 - - 3 4 8 Magnusson, R.P. and McCarty, R.E. (1976) Biochem. Biophys. Res. C ommun. 70, 1283--1289 Oliver, D. and Jagendorf, A.T. (1976) J. Biol. Chem. 251, 7 1 6 8 - - 7 1 7 5 Boyer, P.D., Cross, R.L. and Momsen, W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2837---2839 Boyer, P.D. (1975) FEBS Lett. 50, 91--94 Harris, D.A., Radda, G.K, and Slater, E.C. (1977) Biochim. Biophys. Acta 4 5 9 , 5 6 0 - - 5 7 2 Boyer, J.S. and Knipling, E.B. (1965) Proc. Natl. Acad. Sci. U.S. 54, 1044--1051 Lien, S. and Racker, E. (1971) Methods Enzymol. 2 3 , 5 4 7 - - 5 5 5 Dilley, R. (1972) Methods E n z y m o l . 24b, 68--74 Younis, H.M., Winget, G.D. and Racker, E. (1977) J. Biol. Chem. 252, 1814--1818 Arnon, D.I. (1949) Plant Physiol. 24, 1--5 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 Warburg, O. and Christian, W. (1941) Biochem. Z. 310, 384 Farron, F. and Racker, E. (1970) Biochemistry 9, 3829 3836 McCarty, R.E. (1971) Methods Enzymol. 23, 251--253 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 Weber, G. and Bablouzian, B. (1966) J. Biol. Chem. 241, 2558 2561 Jameson, D.M., Spencer, R.D., Mitchell, G. and Weber, G. (1979) Rev. Sci. Instrum., in the press VanderMeulen, D.L. and Govindjee (1977) Eur. J. Biochem. 78, 585~-598 Weber, G. (1952) Biochem. J. 5 1 , 1 4 5 - - 1 5 5 Laurence, D.J.R. (1969) in Physical Methods in Macromolecular Chemistry (Carrol, B., ed.), pp. 275--351, Marcel Dekker, New York, NY Weber, G. (1952) Biochem. J. 5 1 , 1 5 5 - - 1 6 7 Greenfield, N. and Fasman, G.D. (1969) Biochemistry 8, 4108---4116 VanderMeulen, D.L. and Govindjee (1975) FEBS Lett. 5 7 , 2 7 2 - - 2 7 5 Shahak, Y., Chipman, D.M. and Shavit, N. (1973) FEBS Lett. 33, 293--296 Gixault, G. and Galmiche, J.M. (1976) Biochem. Biophys. Res. C ommun. 68, 724--729 Laurence, D.J.R. (1952) Biochem. J. 5 1 , 1 6 8 - 1 8 0 Jagendorf, A.T. (1972) Methods Enzymol. 24b, 103--113 Gr~oer, P., Sehlodder, E. and Witt, H.T. (1977) Biochim. Biophys. Acta 461, 426--440 Boyer, J.S. (1971) Plant Physiol. 4 7 , 8 1 6 - -820 Potter, J.R. and Boyer, J.S. (1973) Plant Physiol. 5 1 , 9 9 3 - - 9 9 7 Lien, 8. and Racker, E. (1971) J. Biol. Chem. 246, 4 2 9 8 - - 4 3 0 7 Berzborn, R.J. and Schr~er, P. (1976) FEBS Lett. 70, 271--275 Racker, E. (1977) Annu. Rev. Biochem. 46, 1 0 0 7 - 1014 Boyer, J.S. (1973) P h y t o p a t h o l o g y 6 3 , 4 6 6 - - 4 7 2
Biochimica et Biophysica Acta, 5 4 8 ( 1 9 7 9 ) 3 2 8 - - 3 4 0 © Elsevier/North-Holland
Biomedical Press
BBA 47747
CONFORMATION AND ACTIVITY OF CHLOROPLAST COUPLING FACTOR EXPOSED TO LOW CHEMICAL POTENTIAL OF WATER IN CELLS
H A S S A N M. Y O U N I S *, J O H N S. B O Y E R * * a n d G O V I N D J E E
Departments of Botany, Agronomy, and Physiology and Biophysics and United States Department of Agriculture, Science and Education Administration, Agricultural Research, 289 Morrill Hall, University of Illinois, Urbana, IL 61801 (U.S.A.) (Received November 10th, 1978) (Revised manuscript received April 10th, 1979}
Key words: Photophosphory lation; Coupling factor; Chemical potential; A TPase ; Conformation; Nucleotide binding
Summary (1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (~w) of--25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low xPw had less Ca2+-ATPase activity than control CFI and did not recouple phosphorylation in CF1deficient chloroplasts. In contrast, CF~ from leaves having high ~w only partially recoupled phosphorylation by CFrdeficient chloroplasts from leaves having low ~w. This indicated that low ~w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low ~w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that * Present address: D e p a r t m e n t of Plant Protection, Faculty of Agriculture, and Biochemistry and Molecular Biology, University Science Center, Univeristy of Alexandria, Egypt. ** Address reprint requests to J.S. Boyer, USDA/SEA/AR, D e p a r t m e n t of Botany, 289 Morrll Hall, University of Illinois, Urbana, IL 6 1 8 0 1 , U.S.A. Abbreviations: ~ w , water potential CF l , coupling factor protein (ATP synthetase when attached to the membrane); e-ADP, 1,N6-ethenoadenosine diphosphate; e-ATP, 1,N6-etlienoadenosine triphosphate; EDTA, ethylenediaminetetraacetic acid; Tricine, N-tris(hydroxymethyl)methylglycine.
329 CFI from leaves having low ~w differed from control CF~. The CF~ from leaves having low ~w also had decreased ability to bind fluorescent nucleotides (e-ATP and e-ADP). (4) Exposure of isolated CF~ to low ~/w in vitro by preincubation in sucrosecontaining media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2÷ in the preincubation medium markedly inhibited Ca2÷-ATPase activity. (5) These results show that CF~ undergoes changes in cells which alter its phosphorylating ability. Since low cell ~/w changed the spectroscopic properties but not other protein properties of CFI, the changes were most likely caused by altered conformation of the protein. This decreased the binding of nucleotides and, in turn, photophosphorylation. The inhibition of ATPase activity in CF~ in vitro at low ~w and high ion concentration mimicked the change in activity seen in vivo.
Introduction
The chemical potential of water (hereafter described in terms of the water potential, ~w) * decreases when cell water content decreases [1]. In the chloroplast-containing cells of plants, the decrease is associated with large losses in chloroplast activities, notably electron transport and photophosphorylation [2--5]. The losses occur at ~w that are commonly encountered under natural conditions [2,3] and may account for some of the decrease in rates of photosynthesis observed at low ~w [2,5]. Fellows and Boyer [4] presented evidence that the inhibition was correlated with changes in the thickness of the lamellar membranes of the thylakoids in energized chloroplasts. The changes in thickness could be seen in vivo and persisted in vitro. However, the nature of the membrane structure that accounts for the changes in thickness has not been identified. In this paper, we present evidence that the molecular basis of these changes is attributable in part to changes in the conformation of chloroplast coupling factor (CF1). Structural transformations of CF~ have been demonstrated by the ability of the protein to trap tritiated water [6,7] and to bind N-ethylmaleimide [ 8,9], adenine nucleotides [ 10--12], and trinitrobenzenesulfonate [13] when the thylakoid membranes are energized in vitro. With the demonstration that CFl-bound nucleotides undergo a light-dependent and uncouplersensitive exchange with free nucleotides [11,14], it has been proposed that the structural transformations are conformational changes in CF1 that induce changes in the binding affinities for ADP, Pi, and ATP during photophosphorylation [15,16]. Results in the present work indicate that changes in CFI conformation also occur within cells, persist after isolation, and lead to altered
* T h e w a t e r p o t e n t i a l is r e l a t e d t o t h e c h e m i c a l p o t e n t i a l 0~, e r g s • m o 1 - 1 ) o f w a t e r a c c o r d i n g t o xP w ffi (~uw - - / ~ 0 ) / V , w h e r e t h e subscripts w a n d 0 r e f e r t o t h e s y s t e m and the r e f e r e n c e , r e s p e c t i v e l y , a n d V is t h e p a r t i a l m o l a l v o l u m e o f l i q u i d w a t e r ( c m 3 • t o o l -1 , v i r t u a l l y a c o n s t a n t o v e r t h e range o f xIJw i n b i o l o g i c a l s y s t e m s ) . F o r c o n v e n i e n c e , w e r e p o r t ~ w i n p r e s s t t r e u n i t s , 1 b a r ffi 1 0 6 e r g s • c m - 3 = 0 . 9 8 7
atm.
330 affinities for nucleotides that may account for alterations in phosphorylating activity. Materials and Methods
Plant material and ~w. Commercial spinach (Spinacea oleracea L.) was purchased from a local market. Different leaf ~w were obtained by partial drying of the leaves for 1-.--3 h in the light in a controlled environment room (temperature = 29°C; relative humidity = 40--60%; irradiance = 150 W" m -~ from fluorescent (daylight) bulbs). Leaf qJw was measured with a thermocouple psychrometer by the isopiestic technique described by Boyer and Knipling [17]. Measurement o f chloroplast activities. Chloroplasts were isolated from the leaves as described by Lien and Racker [18]. Chloroplast Ca2+-ATPase was activated by heat at 6 0 6 4 ° C for 4 min in the presence of ATP (20 mM) and dithiothreitol (5 mM), and chloroplast Mg2+-ATPase was activated by incubation with dithiothreitol (25 mM) for 15 min. Activities of ATPase were measured as described by Lien and Racker [18] for Ca2+-dependent ATPase, and by replacing Ca 2÷ with 5 mM MgC12 for Mg2+-dependent ATPase. Phenazine methosulphate-mediated cyclic photophosphorylation was measured potentiometrically with a recording pH meter as described by Dilley [19]. Illumination was with saturating (180 W • m -2 between 400 and 700 nm) red light (maximum irradiance at 668 nm), passed through a heat filter [5]. Coupling factor was prepared by extraction with chloroform according to Younis et al. [20]. The protein was stored at 4°C in 2 M (NH4)2SO4, 2 mM ATP, 1 mM EDTA, and 20 mM Tricine/OH-, pH 8, until needed for measurements. Prior to use, the suspension was centrifuged (14 000 × g), resuspended in 40 mM Tricine/OH-, pH 8, recentrifuged (14 000 ×g), and desalted on a Sephadex G-50 column (20 × 1 cm). This procedure provided a protein of high specific activity relatively free of other proteins (Ref. 20, and see Fig. 3 below). The Ca2÷-ATPase of the protein was heat activated at 6 0 - 6 4 ° C for 4 min and assayed as for the chloroplasts. Chlorophyll was determined spectrophotometrically in 80% acetone extracts [21]. Protein concentration was measured usually by the m e t h o d of Lowry et al. [22] and sometimes spectrophotometrically [23]. In the former method, the concentration was multiplied by 1.15 and, in the latter, by a factor of 1.85 to express the protein concentration on a dry weight protein basis [24]. For preparation of CFl-depleted chloroplasts, the chloroplasts were treated with EDTA as described by McCarty [25]. Phosphorylation was reconstituted in these chloroplasts by adding CF1, and phenazine methosulfate-mediated cyclic photophosphorylation was assayed as described in Table II. Polyacrylamide gel electrophoresis of the protein was performed as described by Weber and Osborn [26]. Fluorescence measurements. Fluorescence polarization of CF1 was measured with a photon-counting polarization p h o t o m e t e r in the laboratory of G. Weber. This instrument (see Weber and Bablouzian [27] and Jameson et al. [28]) allows simultaneous monitoring of the parallel and perpendicular components of the polarized fluorescence by using two photomultipliers at right angles to
331 the exciting beam. It also permits convenient subtraction of background scattering at low signal-to-noise values. The excitation wavelength was 280 nm and the emitted fluorescence was filtered through Coming * glass C.S. 0-54 filters. The binding of fluorescent nucleotides (e-ADP and e-ATP) to CFI was followed b y measuring the polarization of the average free and b o u n d e-nucleottide fluorescence as a function of e-nucleotide concentration [29]. The excitation wavelength was 310 nm (passed through a Coming glass C.S. 7--54 filter) and the emission at 405 nm (filtered through a Coming glass C.S. 3-75 filter) was measured. Circular dichroism measurements. The circular dichroism spectrum of CF~ in 20 mM Tricine/OH-, pH 8.0, was measured between 200 and 250 nm with a JASCO model J 4 0 A Automatic Recording Spectropolarimeter. The spectropolarimeter was calibrated with D-(+)-camphorsulfonic acid; the pathlength was 1 mm; the protein concentration was 0.5 mg • m1-1, and the temperature was 25°C. The mean residue molecular weight was taken to be 114. Ultraviolet spectra. Ultraviolet absorption spectra of the protein were measured in an Aminco s p e c t r o p h o t o m e t e r DW-2. Results The photophosphorylating activity of spinach chloroplasts decreased with decreasing leaf q]w (Fig. 1). In chloroplasts from leaves with a q z of a b o u t - 2 5 bars, the rate of cyclic photophosphorylation was 45% of that in the control chloroplasts. A similar result was found with sunflower leaves, in which photophosphorylation was markedly inhibited and uncoupled from electron flow at low ~w [3]. To determine whether the effect involved the phosphorylating enzyme CF1 or other aspects of phosphorylation, we assayed for Ca2÷-depen dent and Mg2*-dependent ATPase activity of the chloroplasts. There was a linear decrease in the Ca2*-ATPase activity of the chloroplasts as leaf q]w decreased (47% of control at ~ w of --25 bars, Fig. 2, solid circles). The Mg 2÷dependent ATPase activity also decreased with decreasing ~w (Fig. 2, open squares). The Ca2÷-ATPase activity recovered fully if the leaves were rehydrated before the chloroplasts were isolated (Fig. 2, open circle). The above results suggest that some of the effects of low q]w on photophosphorylation were caused by an effect on CF~ itself. Therefore, we studied CF~ from leaves with various ~ . Table I shows that CF1 isolated from chloroplasts of leaves having t w o different ~w yielded the same a m o u n t of CFI protein b u t had different Ca2÷-ATPase activities. Activity of the protein from leaves with low ~w was much lower (23% of control) than for protein from leaves with high ~w when the ATPase was n o t heat activated (Table I). However, the relative difference in activity was reduced after heat activation (Table I). Since the protein was probably closest to the in vivo form before heat activation, we
o f a t r a d e m a r k or p r o p r i e t a r y p r o d u c t d o e s n o t c o n s t i t u t e a g u a r a n t e e o r w a r r a n t y o f t h e p r o d u c t b y t h e U . S . D e p a r t m e n t o f A g r i c u l t u r e and d o e s n o t i m p l y its approval t o t h e e x c l u s i o n o f o t h e r p r o d u c t s that m a y a l s o b e s u i t a b l e .
* Mention
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Leaf Water Potentiat (bars)
Leaf Water Potential (bars}
F i g . 1. P h o t o p h o s p h o r y l a t i n g a c t i v i t y o f c h l o r o p l a s t s i s o l a t e d f r o m s p i n a c h (S. oleracea L.) leaves w i t h various w a t e r potentials. Control chloroplasts were isolated f r o m leaves of the s a m e p o p u l a t i o n w i t h o u t d e s i c c a t i o n . T h e c o n t r o l r a t e w a s 1 0 6 0 # t o o l A T P " h -1 • m g - l Chl, p h e n a z i n e m e t h o s u l f a t e c o n c e n t r a t i o n w a s 15 # M a n d c h l o r o p h y l l c o n c e n t r a t i o n w a s 20 # g • m1-1 . F i g . 2. T h e C a 2 + - A T P a s e ( e ) a n d M g 2 + - A T P a s e (~) a c t i v i t i e s o f c h l o r o p l a s t s i s o l a t e d f r o m s p i n a c h l e a v e s with various water potentials. Mg2+-ATPase was activated with dithiothreitol. Recovery from low water P o t e n t i a l (o) s h o w s t h e a c t i v i t y o f C a 2 + - A T P a s e a f t e r r e h y d r a t i o n o f t h e t i s s u e f r o m a w a t e r p o t e n t i a l o f --24.5 bars.
used CF1 t h a t had n o t been heat activated during the following measurements, except where stated.
Coupling activity of the isolated proteins Table II summarizes the results of an experiment in which chloroplasts from leaves with high and low ~w were depleted of CF~ by EDTA treatment, then
TABLE I Y I E L D A N D Ca2+-ATPase A C T I V I T Y O F C O U P L I N G F AC T OR ISOL AT E D FR OM C HL OR OPL AST S OF LEAVES WITH HIGH (CONTROL) AND LOW WATER POTENTIALS E x p e r i m e n t a l c o n d i t i o n s as i n M a t e r i a l a n d M e t h o d s . Treatment
Control Water deficient
Leaf water potential (bars)
---3 --17
Total chlorophyll (mg • extract -I )
15.8 15.2
Total protein (rag extract -I )
8.8 8.9
Protein released/ chlorophyll ( m g • m g -1 )
0.55 0.58
Ca2 +-ATPase a c t i v i t y ~umol Pi " m i n - I " m g - I protein) Before heat activation
After heat activation
2 0.46
14 11
333 T A B L E II R E C O N S T I T U T I O N OF P H O T O P H O S P H O R Y L A T I O N IN C O U P L I N G F A C T O R - D E F I C I E N T CHLOR O P L A S T S BY C O U P L I N G F A C T O R F R O M L E A V E S W I T H H I G H ( C O N T R O L ) A N D L O W W A T E R POTENTIALS P h o s p h o r y l a t i o n was r e c o n s t i t u t e d b y c o m b i n i n g t h e E D T A - t r e a t e d c h l o r o p l a s t s w i t h 1 m l o f t h e i r s u p e r n a t a n t ( c o n t a i n i n g CF l ) to give a c h l o r o p h y l l c o n c e n t r a t i o n o f 0 . 2 r n g / m l . MgCl 2 ( 1 0 m M ) was a d d e d a f t e r the E D T A - t r e a t e d c h l o r o p l a s t s h a d b e e n a d d e d a n d t h e m i x t u r e w a s i n c u b a t e d f o r 15 rain b e f o r e assay at r o o m t e m p e r a t u r e . A t t h e s a m e t i m e , a l i q u o t s o f t h e u n t r e a t e d c h l o r o p l a s t s were d i l u t e d to t h e c h l o r o p h y l l c o n c e n t r a t i o n of t h e t r e a t e d s a m p l e s . P h o s p h o r y l a t i n g a c t i v i t i e s w e r e meastLred w i t h 10 p g c h l o r o p h y l l / m l in a 2 m l r e a c t i o n m i x t u r e c o n t a i n i n g : 3 m M N a H 2 P O 4, 3 m M MgC12, 17 m M KC1, 1.5 m M A D P a n d 5 0 pM p h e n a z i n e m e t h o s u l f a t e , p H 7.8. P r o t e i n c o n c e n t r a t i o n in b o t h s u p e r n a t a n t s was 0 . 1 4 m g / m l as d e t e r m i n e d b y t h e m e t h o d o f L o w r y et al. [ 2 2 ] . Treatment
1. U n t r e a t e d c h l o r o p l a s t s f r o m leaves w i t h a ~Isw o f a. --1 b a r ( c o n t r o l s ) b. - - 1 8 b a r s 2. E D T A - t r e a t e d c h l o r o p l a s t s f r o m leaves w i t h a ~ w o f a. --1 b a r ( c o n t r o l s ) b. - - 1 8 b a r s 3. E D T A - t r e a t e d c h l o r o p l a s t s f r o m l e a v e s w i t h a ~ w o f --1 b a r ( c o n t r o l s ) plus a. CF 1 f r o m leaves w i t h ~ w o f --1 b a r b . CF 1 f r o m leaves w i t h ~ w o f - - 1 8 b a r s 4. E D T A - t r e a t e d c h l o r o p l a s t s f r o m l e a v e s w i t h ~ w o f - - 1 8 b a r s plus a. CF 1 f r o m leaves w i t h ~ w o f --1 b a r b. CF 1 f r o m leaves w i t h ~ w o f - - 1 8 b a r s
Cyclic p h o t o phosphorylation (pmol ATP • h -1 • m a -1 Chl)
1060 440 460 160 700 400 220 170
reconstituted by adding back the CFl-containing supernatant. The CFI released from leaves with low ~w did not restore phosphorylation in CFl-depleted chloroplasts from control leaves (Table II, cf. lines 2a, 3a and b). This result indicates a specific effect of low ~w on CF1. However, control CFI restored phosphorylation only slightly in CF~-depleted chloroplasts from leaves with low ~w (Table II, cf. lines 2b, 4a and 3a). Therefore, in addition, to a specific effect on CF~, low ~w also altered the membrane.
Electrophoretic mobility and subunit structure Coupling factor placed on polyacrylamide gels exhibited a single band with a mobility unaltered by ~w (RF of 0.43 for CFI from leaves with ~w o f - 2 and --18 bars). When detergent was added to the gels to separate the protein subunits, five subunits were always present (Fig. 3), each with an R F unaffected by ~w.
Polarization of CF1 fluorescence The lifetime of the excited state o f fluorescence in molecules is o n the order of 10 -8 s [ 3 0 , 3 1 ] . Because the relaxation times for the rotation o f fluorescent macromolecules are of the same order o f magnitude, their emitted fluorescence is partially polarized, and the degree o f polarization affords a convenient means of determining their relaxation time under a variety o f circumstances [ 3 0 , 3 2 ] . In identical media, and with the assumption that the lifetime o f the excited
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Fig. 3. Densitometrie scans along polyacrylamide gels showing the subunits of (a) CFI isolated from s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f ---2 bars a n d (B) CF 1 i s o l a t e d f r o m s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f - - 1 8 b a r s . T h e p r o t e i n s u b u n i t s (~ - - e) w e r e s e p a r a t e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s in t h e p r e s e n c e o f s o d i u m d o d e c y l sulfate, Gels w e r e l o a d e d w i t h 50 #g o f p r o t e i n ( A ) or 70 # g o f p r o t e i n (B) in 4 0 m M T r i e i n e / O H - , p H 8, b u f f e r . Fig. 4. Circular d i c h r o i s m s p e c t r a o f c o u p l i n g f a c t o r i s o l a t e d f r o m s p i n a c h leaves w i t h w a t e r p o t e n t i a l s of - - 2 . 9 b a t s or - - 1 8 . 7 bars. T h e p a t h l e n g t h was 1 t a r a , the p r o t e i n c o n c e n t r a t i o n w a s 0 . 5 rag • m l - ! • a n d t h e b u f f e r was 20 m M T r i c i n e / O H - , p H 8.0.
state is constant, differences in polarization of a particular substance are caused by differences in conformation. Table III shows that the degree of polarization of fluorescence in CFI from leaves with low ~w (--16 bars) was less by about 0.036 (+0.003) than in CFI from the control leaves (--1 bar). Circular dichroism spectra In the ultraviolet region between 200 and 250 nm, circular dichroism spectra are uniquely determined by protein conformation. Although a number of properties of the protein contribute to the spectra in this region, the quantity
TABLE III FLUORESCENCE POLARIZATION OF COUPLING FACTOR PROTEIN ISOLATED FROM SPINACH LEAVES WITH DIFFERENT WATER POTENTIALS T h e p r o t e i n was d e s a l t e d o n a S e p h a d e x G - 5 0 ( 2 0 × 1 c m ) c o l u m n . T h e b u f f e r was 4 0 m M t r i c i n e / O H , p H 8. P r o t e i n in the s a m e b u f f e r at a c o n c e n t r a t i o n o f 0 . 4 2 m g / m l was u s e d f o r m e a s u r i n g t h e p o l a r i z a t i o n o f f l u o r e s c e n c e as d e s c r i b e d in Materials a n d M e t h o d s . D e g r e e of p o l a r i z a t i o n is F I I - F.L/FI[ + F±, w h e r e FI[ a n d FJ. are t h e f l u o r e s c e n c e i n t e n s i t y parallel a n d p e r p e n d i c u l a r , r e s p e c t i v e l y , t o t h e e x c i t i n g r a d i a t i o n . I n s t r u m e n t f l u c t u a t i o n s i n t r o d u c e e r r o r of ~ 0 . 0 0 3 in p o l a r i z a t i o n values. Water potential of leaves (bars) --1
--16
Degree of polarization of fluorescence by isolated coupling factor
0.306 0.270
335
of s-helix, the orientation of secondary (~) structure, and the arrangement of 'randomly' coiled regions of the protein are particularly contributory. For CF1 from leaves having ~w of --2.9 and --18.7 bars, the molecular ellipticity (0) differed in the same buffer and at the same protein concentration (Fig. 4). Both proteins exhibited a similar extremum around 208 nm, which indicates that both contained a similar amount of s-helix (21% as calculated from Greenfield and Fasman [33]). However, CFI from leaves with ~w of--18.7 bars had a smaller molecular ellipticity between 208 and 230 nm than CF, from control leaves, which suggests that ~ structure and randomly coiled regions had been altered by treatment at low ~w [33].
Ultraviolet absorption spectra The CF, isolated from leaves with ~w of --17 bars absorbed less in the ultraviolet region of the spectrum than did CF1 from leaves with high *w (Fig. 5). The differences in the spectra peaked at 230 and 271 nm {Fig. 5, inset). Because these differences occurred in proteins having the same number of subunits each having essentially the same mobility on polyacrylamide gels, the amino acid composition of CF, was unlikely to have been substantially altered by ~w treatment. Therefore, the differences in spectra were probably caused
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210 230 250 270 290 310 .330 350 Wovelength (nm) Fig. 5. U l t r a v i o l e t a b s o r p t i o n s p e c t r a o f c o u p l i n g f a c t o r f r o m s p i n a c h leaves w i t h a w a t e r p o t e n t i a l o f ( A ) - - 2 bars a n d (B) - - 1 7 bars. Inset: T h e s p e c t r a l d i f f e r e n c e b e t w e e n ( A ) and (B). T h e l o w e s t trace in the m a i n figure is f o r t h e b u f f e r . T h e p r o t e i n c o n c e n t r a t i o n w a s 1.2 r n g / m l in 4 0 m M T r i c i n e / O H - , p H 8.
336
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Fig. 6. P o l a r i z a t i o n o f e - A T P a n d e - A D P f l u o r e s c e n c e b y c o u p l i n g f a c t o r a t v a r i o u s e - A T P a n d e - A D P c o n c e n t r a t i o n s . ( A ) e - A T P plus c o u p l i n g f a c t o r p r o t e i n f r o m c o n t r o l leaves w i t h w a t e r P o t e n t i a l of --2 bars ( e ) o r - - 2 5 b a r s (o). Inset: e - A T P plus c o u p l i n g f a c t o r (a s e p a r a t e p r e p a r a t i o n ) f r o m c o n t r o l leaves w i t h w a t e r p o t e n t i a l o f --2 bars (m) o r - - 1 7 bars ( a ) . (B) e - A D P plus c o u p l i n g f a c t o r f r o m leaves w i t h w a t e r p o t e n t i a l o f --2 bars ( c o n t r o l ) ( e ) o r - - 2 5 bars (o). T h e m e d i u m c o n t a i n e d 1.4 # M p r o t e i n a n d 40 m M T r i c i n e / O H - , p H 8.
TABLE IV E F F E C T O F W A T E R P O T E N T I A L A N D Mg 2+ ON H E A T - A C T I V A T E D Ca2÷-ATPase A C T I V I T Y O F COUPLING FACTOR T h e p r o t e i n f r o m l e a v e s w i t h a high ~ w w a s i n c u b a t e d for 30 m i n b e f o r e t h e a s s a y at r o o m t e m p e r a t u r e ( A ) b e f o r e h e a t a c t i v a t i o n or (B) a f t e r h e a t a c t i v a t i o n . I n p r e p a r a t i o n f o r i n c u b a t i o n , a s a m p l e o f a m m o n i u m sulfate s u s p e n s i o n o f CF 1 was c e n t r i f u g e d a n d t h e p e l l e t was dissolved in 4 0 m M T r i c i n e / O H - . p H 8, a n d r e c e n t r i f u g e d a g a i n a t 14 0 0 0 X g f o r 10 re.in. T h e c l e a r s u p e r n a t a n t was d e s a l t e d o n a 20 × 1 c m Sep h a d e x G - 5 0 c o l u m n . S u c r o s e was a d d e d as solid sucrose a n d MgC12 was a d d e d f r o m a 1 M s t o c k s o l u t i o n . A f t e r t h e i n c u b a t i o n , Ca2+-ATPase a c t i v i t y was d e t e r m i n e d a f t e r a d d i t i o n of 20 #l o f t h e i n c u b a t i o n m i x t u r e t o 1 m l o f t h e assay m e d i u m . T r e a t m e n t ( c o m p o n e n t added to p r o t e i n in 4 0 m M T r i c i n e / O H - , p H 8)
None 0.8 M 5 mM 0.8 M 10 mM
sucrose MgCI 2 s u c r o s e + 5 m M MgC12 MgCl 2
0.8 M suCrose + 10 mM MgCI 2
Ca2÷-ATPase a c t i v i t y ( # m o l Pi " m g -1 p r o t e i n • rain - 1 ) (A) T r e a t m e n t b e f o r e heat activation
(B) T r e a t m e n t a f t e r heat activation activation
20 28 21 13 13
14 7 13
--
5 3
337 by a difference in orientation of ultraviolet-absorbing groups on, or light scattering by, the proteins. Binding o f e-ADP and e-ATP Both e-ADP and e-ATP are fluorescent and bind to CF, [29,34]. They can substitute to a considerable degree for ADP and ATP in many reactions in chloroplasts and isolated CF, [29,34--36]. We tested the ability of CF, from leaves with low ~I'w to bind e-ATP and e-ADP by the technique of fluorescence polarization. The fluorescence of these analogs in aqueous solutions, as with many small fluorescent molecules, is n o t polarized because their rotational relaxation times are considerably shorter than their excited lifetimes (approx. 20 ns). The relaxation time is increased by placing the fluorescent molecule in a viscous medium or by attaching the molecule to a macromolecule such as a protein [30,32,37]. As a result, the fluorescence of the adsorbed molecule is partially polarized. Therefore, in any mixture of small fluorescent molecules with a protein, the fraction of the total fluorescence that is polarized will be proportional to the fraction of fluorescent molecules b o u n d to the protein. Under otherwise constant conditions, differences in the binding properties of proteins saturated with the fluorescent molecule represent differences in relaxation times attributable to differences in protein conformation. In the case of e-ADP and e-ATP, differences in binding properties of CF, should reflect differences around the active or regulatory sites for photophosphorylation. Fig. 6 shows the saturation binding of e-ATP and e-ADP to CF, from leaves having various ~I'w in the same buffer and at the same protein concentration. Unlike CF, from leaves with a ~I,~ of --2 bars, which b o u n d large amounts of e-ATP (Fig. 6A), CF, from leaves with a xI,~ of --17 bars b o u n d less e-ATP (Fig. 6A, inset). The dramatic decrease in fluorescence polarization could n o t be attributed to fluorescence quenching by CF,, since the total fluorescence of the control and treated CF, was the same. At xI'w of --25 bars, CF, appeared unable to bind significant e-ATP or e-ADP (Fig. 6). Effects o f Mg 2+ and low ~I'w in vitro on A TPase activity o f isolated CF, During water loss from cells, the concentration of ions around CF, should increase (at a xI'w of --25 bars, the water content of the tissue had decreased to a b o u t 1/3 that in the controls). To test whether prior incubation of CF, in high concentrations of ions could alter the subsequent biological activity of the protein, we preincubated CF, in 5 mM or 10 mM Mg 2+. Preincubation in 5 mM Mg 2+ inhibited the subsequent Ca2+-ATPase activity of CF, only if the CF, had been heat activated (Table IV). At 10 mM, Ca2+-ATPase activity was inhibited regardless of the timing of preincubation in relation to heat activation (Table IV). The direct effect on CF, Of a decrease in the chemical potential o f water was tested b y preincubating CF, in 0.8 M sucrose (~w = - - 2 5 bars). Ca2+-ATPase activity was enhanced if preincubation occurred before heat activation of CF, b u t it was inhibited b y preincubation after heat activation (Table IV). The incubation of CF, in 10 mM Mg 2+ plus 0.8 M sucrose was particularly inhibitory to the Ca2+-ATPase activity of the protein (Table IV). Because the
338 assays measured Ca 2÷ ATPase activity after the incubation, neither sucrose nor Mg2÷ was present in significant quantity during assay. Discussion
These results show that the inhibition of photophosphorylation and ATPase activities of chloroplasts isolated from leaves with low q~w is associated with a change in the spectroscopic properties of coupling factor. Evidence for such a change includes differences in circular dichroism (Fig. 4), polarization of protein fluorescence (Table III), ultraviolet absorption (Fig. 5), and ability to bind nucleotides (Fig. 6). However, there were no significant differences in the yield of protein extractable from the chloroplasts (Table I) or in the number of subunits or the mobility of the protein and its subunits on polyacrylamide gels (Fig. 3). Therefore, the primary structure and molecular weight of the protein were unlikely to have been altered by low ~w, although we cannot exclude the possibility of changes in CF1 binding of other molecules too small to be detected by our gel techniques. With this possible exception, we therefore attribute the spectroscopic differences to changes in the conformation of CF~. Evidence for conformational changes in CF~ has been presented previously for thylakoids energized either by light [6,7], acid-base transitions in the dark [7,13,38], or external voltage pulses [39]. These findings imply that such energy-dependent alterations are required in some way for photophosphorylation. In the present work, CF, was exposed in vivo to low ~w, and the effects on photophosphorylation and conformation persisted after isolation of the protein. Apparently, the conformation of CFI changed in the cells, and the altered conformation was quite stable. Because the effects of photophosphorylation were reversed by rehydration of the cells before isolation of CF~, the altered conformation, although stable, must have been inherently reversible under appropriate conditions in the cells. A similar phenomenon has been seen in chloroplast electron transport; the effect of low ko~ was reversed only if cells were rehydrated before isolation of the plastids [ 5,40,41 ]. The recoupling experiments (Table II) provided a critical confirmation of the effects of low ~Pw on CF,. In these experiments, the protein was unable to restore cyclic photophosphorylation under conditions that readily supported restoration by control CF~. T h e failure of CF~ from low q]w to restore phosphorylation must have been caused by altered biological activity, decreased binding affinity for chloroplast membranes, or both. Clearly, the effect persisted after removal of the protein from the membranes, as had been indicated by the spectroscopic measurements. It is important that control CFI was only slightly able to recouple CF~deficient chloroplasts from leaves with low ~w. Therefore, although low ~w caused a specific effect on CF~, it also altered the chloroplast membranes. Thus, low qJw had multiple effects on chloroplasts, as has been suggested previously by alterations in electron transport and non-cyclic photophosphorylation in chloroplasts from leaves having low ~w [3]. The results (Table II) also add more evidence to what has been discussed by others [42,43] that CF~ added to CF~-deficient chloroplasts has coupling activity in addition to its structural role on the membrane.
339 The most obvious and significant change in CF~ from leaves with a low ~w was the decrease in binding of e-ADP and e-ATP (Fig. 6). It is generally accepted that ATP synthesis involves the binding of ADP and Pi to CF~ and the release of ATP into the medium. Consequently, the marked losses in binding could have contributed to losses in phosphorylating activity at low ~w (Fig. 1). Since concomitant changes in protein conformation also occurred at low q~w, decreased nucleotide binding may have been caused by physical changes in or around the nucleotide binding sites. It should be noted, however, that residual phosphorylation and ATPase activity remained at ~w that markedly reduced binding of fluorescent nucleotides (cf. Figs. 1 and 2 with Fig. 6). The analogs probably bind with less affinity than the normal substrates because of the altered structure of the analogs. In support of this idea, Shahak et al. [35] showed that CFI phosphorylates e-ADP vigorously but the Michaelis constant is about twice that for ADP. The persistence of changes in CF1 after its isolation from cells implies that any attempt to reproduce the effect in vitro must involve a pretreatment of CF~ but assay under uniform conditions to detect effects of the pretreatment. Lowering the water potential of cells increases the concentration of ions in the cytoplasm but also decreases the activity of water molecules. Both effects could bring about conformational changes in CF~. The losses in activity of CF~ after preincubation in high Mg2+ and/or low water activity (Table IV) suggest that CF~ was altered to a different reactive state (Ref. 44 and Younis, H.M., Boyer, J.S. and Govindjee, unpublished results). This new state was stable enough to be subsequently detected by a change in ATPase activity even though the assay was conducted with high water activity and negligible Mg2+. Because the inhibition of ATPase activity in vitro was similar to that in vivo, the in vitro pretreatment seemed to mimic the events occurring within the cells. This conclusion is generally consistent with the behavior of chloroplasts in cells having low ~w. Photosynthetic electron transport and phosphorylation begin to be inhibited 5----10 min [45] after desiccation begins, and recovery after water is resupplied is almost as rapid (15 min [41]). Chloroplast membranes change thickness at the same time but appear normal in other aspects [4]. The rapidity of the changes makes it unlikely that membrane degradation or synthesis is involved and suggest rapid changes in conformation, interaction of membrane subunits, or both. For CF1, the simulation of cell dessication involved rapid changes in water activities and Mg2÷ concentrations over a range likely to occur in cells. The similarity of the simulation and cell dessication results suggest that at least part of the desiccation-induced decrease in the phosphorylating activity of chloroplasts is caused by a simultaneous increase in ion concentration and decrease in chemical potential of water that change the conformation of CF~ and, in turn, decrease the binding of nucleotide substrates to the coupling site(s).
Acknowledgments We are particularly indebted to Dr. Gregorio Weber for help with the fluorescence experiments, the use of his polarization photometer, and for discussions
340
and a critical reading of the manuscript. We also thank Dr. C.J. Arntzen and J. Mullet for assistance in the measurements of ultraviolet absorption spectra. We gratefully acknowledge financial support from the National Science Foundation (J.S.B., PCM 76-11026; G., PCM 76-11657) and the U.S. Public Health Service (G. Weber, GM 11223). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
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