Effect of polyamines on surface charge and light-scattering changes in thylakoid membranes

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Bioelectrochenaistry and Bioenergetics, 32 (1993) 77-81

ElsevierSequoiaS.A.,Lausanne JEC BB 01628 Short communication

Effect of polyamines on surface charge and light-scattering changes in thylakoid membranes Virjinia Doltchinkova a, Dimiter Milkov b and Nadko Naidenov ’ a Department of Biophysics and Radiobiology, Faculty of Biology, Blvd. Dragan Tzankov S, Sofa lJniversi@, Sofia (Bulgaria) ’ L&oratory of Pedagogical Diagnostics, Sojia University, Sofia (Bulgaria) c High Institute for Mechanical and Technical Engineering, Sofa (Bulgaria) (Received 16 May 1992; in revised form 27 January 1993)

INTRODUCTION

Polyamines are widely distributed in the plant kingdom and their biosynthetic pathways are well understood, although their physiological function, particularly in photosynthesis, remains unclear [1,2]. A number of authors have investigated the role of polyamines in the stabilization of membranes. For instance, a stress-mediated increase in diamines (particularly putrescine) has been suggested is an adaptive advantage in plants, and a number of protective functions of polyamines have been proposed [3,4]. Naturally occurring polyamines in Legumineus angiospermae are moderate amount (0.4-0.1 pmol g-r wet weight) [5]. It seems likely that the polybasic nature of polyamines is important in determining their physiological action [6]. The light-scattering (IS) method can be used to detect conformational changes and to characterize the energized state of the thylakoid membrane [7,8]. The increase in LS depends non-linearly on the formation of the light-induced proton gradient. Moreover, the changes in LS at 535 run can be correlated with cation transport under physiological conditions [9]. Coughlan and Schreiber [lo] have concluded that the narrow-angle 90” scattering component is a direct reflection of light-induced proton pumping across the membrane. Measurements of the electrokinetic potential have shown that energization of chloroplast membranes induces a significant increase of negative surface charge density. It has been argued that the net electrical charge density on the outer surface of the grana membranes is low, thus allowing two adjacent membrane surfaces to approach each other closely [11,121. The significant asymmetry of 0302-4598/93/$06.00

0 1993 - Elsevier Sequoia S.A. All rights reserved

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surface charge near the photosystem 2 reaction center [13] could play an important role in the stabilization of charge separation across the membrane [14]. The aim of the present work is to investigate the dependence of changes in the surface charge of thylakoid membranes after PA treatment on their geometry, which is closely related to “stacking” and “unstacking” conditions. We attempt to clarify the probable mechanism of these exogenous polyamine effects, particularly their influence on the kinetic phases of photoinduced LS of thylakoid suspensions. It was found that the effects of primary ionic exchange processes (fast phase of the kinetic curve of photoinduced LS) were greater in thylakoids with polyamines (putrescine, sperimidine or spermine) in the incubation medium, than in controls. MATERIAL AND METHODS

The method proposed by Whatley and Arnon [15] was used to extract thylakoid membranes from 14-day-old pea seedlings (J?.s~rn sutivum L.) which had been grown in a greenhouse under sunlight. The isolation medium contained 67 mM phosphate buffer, 330 mM sorbitol and 5 mM MgSO, (pH 7.80). The chlorophyll concentration was 3.9 mg ml-’ [161. The thylakoids were stored in liquid nitrogen as described by Goldfeld et al. [17]. Immediately prior to use, they were thawed and diluted with an appropriate buffered medium (10 mM N-tris(hydroxymethyl)methylglycine (Tricine), pH 7.80) to a chlorophyll concentration of 6 pg ml-’ where the maximum effect of photoinduced LS was observed. Transfer of isolated thylakoids into a low salt medium (e.g. 10 mM Tricine) led not only to a loss of stacked membrane regions but also to complete intermixing of all intramembrane protein complexes [181. LS of a weak monochromatic beam at wavelength 550 nm was measured at an angle of 90”. Gut-off filters were used to protect the photocell from the actinic light (A 2 640 nm) with intensity 2950 PE mm2 s-l (E denotes einstein). Electrophoretic mobility studies were performed using an OPTON Cytopherometer (FRG). The suspending medium consisted of 10 mM Tricine buffer (pH 7.80). The reaction medium used in the LS experiments contained 40 PM phenazine methosulfate (PMS) which is one of the most effective mediators of photosynthetic electron transport processes. The OPTON Gytopherometer was equipped with a television monitor (Philips) which allowed different types of thylakoid membranes to be observed and migration by 20-40 particles to be timed. Electrophoretic migration was timed for both forward and backward (reversed field) runs over a known distance (80 ELM).The observation light was filtered by a green (545 nm) interference filter. The standard deviations of the mean values of the electrophoretic mobility (EPM) u were of the order of 2%-5%, and never exceeded 10%. The electrical conductance and viscosity of the different media, including the thylakoids, were measured using a Radelkis (Hungary) model, OK-104 conductometer and a Rheo (Germany) viscosimeter respectively. The statistical program was a modification of the least-squares method. Polynomial curve fitting was applied. Photoinduced kinetic LS curves were analysed, and

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Fisher’s F test and Student’s t test were used to determine the significance of the differences. Putrescine dihydrochloride, spermidine trihydrochloride, spermine tetrahydrochloride, Tricine and disodium hydrogen phosphate were purchased from Fluka AG, Switzerland. Potassium dihydrogen phosphate and D-Sorbit DAB 7 were obtained from Loba Chemie Wien-Fischamend, MgSO, was obtained from VEB Jenapharm-Laborchemie Apolda and phenazine methosulfate was obtained from Serva (Germany). RESULTS AND DISCUSSION

Many biological particles, cells and large subcellular structures rapidly change their volume when the medium is changed; this change is accompanied by alterations in LS by their suspensions. An investigation of the dependence of the LS effect on the surface charge has been performed. After 3 min dark adaptation, the photoinduced LS kinetic curve of thylakoids consists of three main phases: fast and slow with different signal amplitudes and characteristic times of enhancement. The intensity of photoinduced LS decreases exponentially after the actinic light is turned off, but does not always return to the initial state. We shall use the term “decay phase” of LS to describe this system. The LS response of a thylakoid membrane to photosynthetically active light (2950 PE mm2 s-‘> is shown schematically in Fig. 1. It can be seen that after a period of fast ionic exchange processes the system reaches a steady state LS,,. After switching off the light the photosynthetic system relaxes back to its original state with a characteristic relaxation time pi,*, i.e. the time required for the “dark” signal to relax to half-maximum amplitude. Hence we can describe the response in terms of three parameters LS,, (fast phase), LSc,,,ax_minj ) (decay phase) relating to the magnitude of LS before (slow phase) and LScmax_stat the light is switched on. Two other parameters are involved in description of the IS kinetic curve: the slope tan (Yof the curve and relaxation time 71/Z. Continuous illumination leads to a net bulk proton influx of magnitude depending on the difference between the turnovers of proton influx and efflux [19]. Photoinduced ionic processes take place in the light and are reflected in the fast phase of photoinduced LS. If the proton pump is electrogenic, a membrane potential is established, leading to accumulation of protons and a change in pH [20]. Thus the energy-dependent proton translocation is related to the secondary ion exchange processes in the slow phase of the kinetic curve. When the light is turned off, the ionic gradients equilibrate as the influx of protons stops immediately [21]. The dark scattering level at 90” represents the degree of stacking [lo], and together with fluorescence yield measurements can be used to monitor the degree of restacking of unstacked thylakoids upon addition of an appropriate ion concentration [22]. The changes in scattering intensity of thylakoids produced by illumination have been interpreted by Thome et al. [23] as being due to changes in

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LIGHT

OFF

I I

LSSTAT

I I I I

2__L&--L_-________~l---___ 5*

4

LIGHT

ON

I

10

min

I

Fig. 1. Characteristic kinetic curve of photoinduced LS formation and decay and parameters derived for analysis: ta, 3 min dark adaptation of thylakoids+PAs; tan a, slope of the curve; LSminr maximum level of the fast ionic-exchange processes; LS,,, steady-state level of the signal; LS,,,, lowest level of the dark relaxation processes; r,,s, time for the decay signal to decrease to half-maximum amplitude. In the LS experiment with dark-adapted membranes, the sample was under constant illumination through KC 13 red and hot filters at an incident light intensity of 2950 PE mm2 s-l. An amplitude resolution equivalent to an intensity change AI/I = 3.5 x 1O-9 was used in the LS measurements.

selective dispersion induced by conformational changes of membrane components caused by protonation of the intrathylakoid luminal space. The technique of particle electrophoresis was used to suspend thylakoid membranes in low ionic strength media (0.0006 M) and their mobility in an externally applied electric field was observed under a microscope. An attempt was made to estimate the surface charge density u from electrophoretic data [11,241. The effect of oligo-cations was tested by measuring the electrophoretic mobility u at pH 7.80

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L I

-5

I

I

I

I

-4

-3

I

-2

Log([POLYAMI NEI/Ml Fig. 2. Electrophoretic mobility of thylakoid membranes suspended in 10 mM Tricine (pH 7.80) treated with putrescine (o), spermidine (A ) and spermine ( n 1. Measurements were performed in the absence of PMS.

in different concentrations of various polyamines. The results show a decrease in u with increasing ionic strength. The effectiveness of polyamines is dependent on their positive charge; electrostatic interactions appear to be a major parameter in their mechanism of action [251. The decrease in the EPM of thylakoid membranes produced by electrostatic interactions was dependent on the valence of the oligo-cations [26], as shown in Fig. 2. The maximum decrease in EPM was determined for 2.8 mM putrescine (PC), 560 PM spermidine (Sd) and 500 PM spermine (Sm) in the suspending medium. We determined the following values of surface charge density u for thylakoid membranes with polyamines in the suspending medium: thylakoid + Pc2+,

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-3001, 0

I

100 SPERMINE

I

200

360

wnmtmtion/pM

Fig. 3. The percent change in the fast (01, slow (A> and decay (B) phases of LS as a function of spermine concentration. The standard reaction mixture contained 10 mM Tricine buffer (pH 7.80) (adjusted with 0.005 M KOH), 40 PM PMS and various spermine concentrations. Other experimental details are as in Fig. 1.

-0.01037 to - 0.00410 C rnp2; thylakoid + Sd3+, -0.01036 to -0.00407 C rne2; thylakoid + Sm4+, - 0.01036 to - 0.00177. The surface charge density is decreased by spermine, spermidine and putrescine in the order Sm4+> Sd3+> Pc2+. The most significant changes in photoinduced LS were produced by spermine. At spermine concentrations above 50 PM in the thylakoid incubation medium, the fast phase of photoinduced LS (which is probably related to ion-exchange processes) decreased by about 260% compared with the control values (Fig. 3). The inhibition of photoinduced LS at lower concentrations of spermine (up to 110 PM) and the highest concentrations of spermidine (390 and 560 PM) (Fig. 4) may be explained by the difficulty of photoinducing ion-exchange processes. The same order of effectiveness of polycations (Sm4+> Sd3+> Pc2+) was observed for the fast phase of photoinduced light-scattering. There is a close relationship between photoinduced ion-exchange processes and the surface charge density value. It is known that even small polyamine concentrations can considerably compensate the negative surface charge of thylakoid membranes [26], which leads to a decrease in the electric gradients of the membrane [27]. Ion-exchange processes in thylakoid membranes are strongly inhibited by spermidine (concentrations above 300 FM) and putrescine (Figs. 4 and 5) in media of low ionic strength. Activation of the processes is highest at spermidine concentra-

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180-

801 , 0

I 100

I 200 SPERMIDINE

I 300 concentration

I 400

I 500

/,uM

Fig. 4. Percentage change in the fast (a), slow (A) and decay Cm) phases of LS as a function of spermidine concentration. The standard reaction mixture contained 10 mM Tricine buffer (pH 7.80) (adjusted with 0.005 M KOH), 40 PM PMS and various spermidine concentrations.

tions of 30-100 ,uM, but decreases to 30% at concentrations of 400-560 PM. The maximum effect on the fast phase of photoinduced LS occurs at a putrescine concentration of 1 mM. The slow phase of photoinduced LS (proton transfer accompanied by the transfer of other ions) was altered by treatment with polyamines. Activation of the slow phase is highest at spermidine concentrations of 30-55 PM (Fig. 4), and at a spermine concentration of 30 PM (Fig. 3) and a putrescine concentration of 1.7 mM (Fig. 5). Spermidine appears to be involved in the modification of ion uptake taking place at the thylakoid proton gradient level; the other polyamines (putrestine and spermine) could regulate ApH to a lesser extent. A well-defined positive correlation was obtained between the increased number of positive charges (PC*+, Sd3+, Sm4+) and enhancement of relaxation time rr,* (data not shown). The photoinduced LS phases of thylakoid membranes with and without polyamines in incubation medium were compared by statistical methods. Statistically significant variations in the fast phase of photoinduced LS were observed only for putrescine and spermine.

a4

8OL 0

1

2

PUTRESCINE concentrotion/mM

Fig. 5. Percentage change in fast (01, slow (A) and decay (m) phases of LS as a function of the putrescine concentration. The standard reaction mixture contained 10 mM Tricine buffer (pH 7.80) (adjusted with 0.005 M KOH), 40 PM PMS and different putrescine concentrations.

Spermine produced 72% stacking in thylakoids at the dark level of IS. Putrestine and spermidine had no effect. No direct relationship was observed between the degree of stacking of the thylakoid membranes and the kinetic curve of photoinduced IS due to polyamine effects. A large decay effect was observed in the fast phase probably because of (9 the increased level of stacking and (ii) the positive charge on spermine which allows two thylakoid membrane surfaces to approach each other more closely. Fewer attractive sites between thylakoid membranes in the suspending medium occurred at lower spermine concentrations. Spermine-treated thylakoid membranes were characterized by a higher negative surface charge density compared with the thylakoids treated with putrescine and spermidine. Higher spermine concentrations in the suspending medium result in almost complete stacking of unstacked thylakoids. As was pointed out above, the thylakoid membranes were suspended in a low ionic strength medium (I = 0.0006 M, 10 mM Tricine). According to Paulus et al. [28], this may reflect the greater binding of polyamines produced by a reduction in ionic strength. Polyamines added at lower concentrations are more freely diffusible and thus lead to marked changes in photoinduced IS kinetic curves as a result of volume rearrangement. Inhibition of primarily ion-exchange processes was observed, as shown by the rapid decrease in the fast phase of photoinduced IS after addition of increasing concentrations of spermine to the suspending medium. Hence the conformation of the protein structures (i.e. thylakoid protein clusters of light-col-

8.5

letting chlorophyll + protein complexes) is changed as a result of electrostatic interactions between adjacent stacking membranes in the presence of spermine. This is evidence that spermine may exert a clamp-like action [29]. LS from thylakoid membranes treated with putrescine and spermidine reaches a maximum at concentrations of 0.56-1.10 mM and 28-110 PM respectively. CONCLUSION

There is little doubt that most polyamine effects are due to their polycationic nature and consequent electrostatic interactions with polyanions [30]. In the gel phase the physical properties of the lipid bilayer change, increasing fluidity and consequently influencing the transport processes 1311 resulting in a P=O.. . H-N hydrogen bond. Thus the polyamines could interact with proteins and with anionic sites on the membranes. It is known that polyamines have a structure in which positive charges are distributed along a flexible chain, which facilitates this interaction. Since PC, Sd and Sm are oligo-cations at physiological pH, they bind to thylakoid membranes and change their permeability, i.e. ion exchange takes place with strongly destacked thylakoid membranes depending on the increasing number of charged centers. We have prepared thylakoids and suggested that polyamines may be involved in the outer surface and/or in interactions with intergranal proteins from the internal surface. Putrescine, spermidine and spermine alter the refractive properties of the membrane surface because of the change in polyamine concentration in the membrane boundary layers. When thylakoid membranes are treated with putrescine and spermidine, an increase in photoinduced LS is observed which is not accompanied by significant stacking processes. Higher concentrations of spermine promote attraction between the charged surfaces due to hydrogen bonds, so that one side of the membrane approaches the other negatively charged side more closely. The neighboring membranes are stacked, and the degree of stacking is related to the increased concentration of spermine in thylakoid suspension. The magnitude of the slow phase (i.e. the proton gradient across the membrane after polyamine treatment) decreases with increasing number of positive charges (PC’+< Sd3+< Sm4+). Thus Sm is the most effective oligo-cation, Sd is less effective and PC has least effect on the ApH gradient. The decrease in the decay phase became more marked as the positive charge on the polyamine increased. The origin of the sharp inhibition of the ion-exchange processes may be a large conformational change in the chlorophyll + protein complexes during pumping. The increase in the stacking level of thylakoid membranes correlates with the decreased value of the negative surface charge density. The addition of oligo-cations to solutions containing thylakoid membranes results in a large reduction in the surface charge density. Proton pumping in light is a result of H-binding to the interthylakoid membrane surface which also decreases the surface charge. A

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dynamic interaction takes place between the electron transport complexes, and the effects of surface charge [24] are important factors in this. Thus the thylakoid volume decreases, i.e. the particle geometry depends strongly on photoinduced IS. The last statement refers to spermine action only, although putrescine and spermidine modify thylakoid membrane structures. Ion-exchange processes are affected by surface charge. However, no well-defined relationship between charge density u and volume changes after polyamine treatment was observed. ACKNOWLEDGEMENTS

We thank Associate Professor V. Goltsev and Professor I. Yordanov for helpful discussions, and M. Gergova and S. Biocheva for technical assistance. REFERENCES 1 2 3 4 5

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