Effect of pyrrolidine dithiocarbamate on photo-induced proton transport through chloroplast membranes

Transcription

1 Vol. 43, No. 5, December 1997 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL Pages Effect of pyrrolidine dithiocarbamate on photo-induced proton transport through chloroplast membranes Edwina Sau-Man Po and John W. Ho* Department of Biochemistry, The Chinese University of Hong kong, Shatin, Hong Kong Received September 17, 1997 Summary ph changes produced by photo-induced proton transport through chloroplast membranes in spinach were measured by a glass microelectrode. Effect of pyrrolidine dithiocarbamate on proton translocation through chloroplast membranes has been studied. Kinetic analysis of proton translocation shows that the rate is reduced as the carbamate concentration increases. The rate of proton uptake follows firstorder kinetics and diminishes with increasing carbamate concentrations. The outward leakage of accumulated protons through thytakoid membranes in the dark also decreases likewise. However, the leakage of protons takes a much longer time. Pyrrolidine dithiocarbamate is an effective inhibitor of proton transport through chloroplast membranes. The results suggest that the photo-induced proton translocation is regulated by conformation change in the membrane. Higher concentration of carbamate disrupts the tertiary conformation of the membrane. The inhibition of proton transport would affect ATPase function; thus, an excess use or accumulation of pyrrolidine thiocarbamate may compromise ATP production. Key words: pyrrolidine carbamate, proton transport, herbicide ntroduction Photosynthesis involves an initial light-dependent decomposition of water, followed by electron transport process in which the reduced and the oxidized fractions recombine to fqrm water. The only net change is the formation of ATP. During ATP hydrolysis protons are translocated from the *Corresponding author /97/ / Copyright by Academic Press Australia. All rights of reproduction in any,/orm reserved.

2 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL stroma into the thylakoid lumen, this results in inhibition of the ATPase activity when the internal proton capacity is reached. On the other hand, the enzyme activity increases upon illumination when the internal proton activity is low. Thus, the observed proton pumping depends on a chemical equilibrium between proton entry and back-diffusion in the dark. Proton transport across the thylakoid membranes is an important process in ATP production. Proton translocation is sensitive to the condition of the membrane and the presence of uncouplers. Proton transport also depends on the chloroplast coupling factor (CF). The rate of proton transport upon activation by light can be measured by the change in ph of the buffer solution using a glass ph electrode [1]. Proton uptake through chloroplast membranes by illuminated chloroplasts is given by the following equation : n ( 1 - ~5/8~s ) = -KL t (1) and the leakage of accumulated protons from chloroplasts in the dark follows the first-order kinetics: n ( 5/Sss ) = -KD t (2) where 8 and ~i,s denote the nmoles proton taken up from the medium per mg chlorophyll at time t and at the steady state; KL and KD are the proton uptake and leakage rate constants, respectively. The rates obtained under illumination could vary depending on the activation of chloroplast coupling factor 1 (CF. The initial rate (R) of proton transport is equal to KL 5,~ [1]. Proton translocation could be inhibited by, for example trypsin [2], octylglucoside [3] and methanol [4]. Weak bases such as pyridine and imidazole were found to increase the rate of electron transport and steadystate proton uptake by illuminated chloroplast membranes [5]. Permeant organic bases accumulated inside the illuminated chloroplasts and decreased the change in ph (A ph). Consequently, it decreases the rate of photophosphorylation but increases the basal rate of electron transport. Other studies indicate that hydrophobic pyridine homologues can bind to 968

3 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL specific sites in the inner membrane and increase the basal rate of electron transport [6]. The kinetic measurement of proton transport in chloroplasts can provide useful information on the fundamental process of proton transport in chloroplasts. n this study, effects of pyrrolidine dithiocarbamate on proton transport in illuminated chloroplast membranes from spinach chloroplasts are examined. The kinetics of the proton translocation through chloroplast membranes are also studied. Experimental Materials Pyrrolidine dithiocarbamate, Tricine, KC1, MgC12, sucrose and NaC1 were purchased from Sigma Chemicals (MO, USA). Acetone and other solvents of analytical grade were purchased from AnalaR BCH Laboratory Supplies (England). Spinach was obtained from local markets. Preparation of chloroplast membranes Chloroplasts were prepared from spinach leaves using the procedures of McCarty with modifications [7]. Deveined leaves were homogenized in a medium containing.25 M sucrose, 2 mm Tricine-NaOH (ph 7.6), 2 mm NaC1. The volume of buffer used for blending was about 6 ml for each gram of leave prepared. The homogenate was filtered through four layers of cheesecloth and then centrifuged at 1,2 x g for 2 min at 4 ~ The chloroplasts were resuspended and homogenized in 5 to 1 ml of Tricine- NaOH buffer prior to storage at 4 ~ Fresh chloroplasts were usually prepared every other day. The stored chloroplasts were re-centrifuged at 1,5 x g for 5 rain prior to experiments. The pellets were resuspended in 1 ml of 2.7 mm Tricine buffer containing.1 M NaC1 and 2.3 mm sucrose before use. The chloroplast preparation was stored in an ice bucket until use. The chlorophyll content is determined according to the Arnon's method [8]. Briefly, an aliquot of 125 gl chloroplast suspension was diluted to 25 ml with 8 % acetone solution. The optical density of the suspension was measured at 663 nm. The concentration was expressed as mg chlorophyll / ml chloroplast suspension. Assay of proton transport Experimental conditions were essentially based on those described earlier [1]. A standard assay medium contained 2 mm Tricine buffer (ph 6.2) at ambient temperature,.1 M NaC1 in a volume of 6. ml. The chlorophyll concentration for each assay was.8 rag. An acidic buffer facilitated a greater change in ph (ApH). Experiment was started by putting 969

4 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL chloroplast suspension in the lab-built glass container with a water-cooling outfit. The whole apparatus was housed in a cardboard box. The chloroplast suspension was allowed to stand in the dark for about 2 rain before illumination of the sample through a small opening in the box with the 3-watt projector light source. The ph change was measured with a glass microelectrode (5 mm dia x 12 ram) obtained from Hanna (taly) connected with an integrator model HP 3395 (Alto Palto, USA). When the ph change reached a plateau, the light was turned off. Once the signal returned to the baseline, titration with 5 gl of.1 M HC into the chloroplast suspension was performed. The proton concentration and the kinetic analysis were carried out according to the previous methods [1]. Results nhibition of proton translocation by carbamate A control experiment without carbamate was treated as 1 % in proton translocation activity. All experiments were performed in at least duplicate. A wide range of carbamate concentrations were used in the study. Figure 1 shows that pyrrolidine carbamate produces a gradual decrease in proton transport in a non linear fashion with its concentration. Time-course study of pyrrolidine dithiocarbamate Effects of carbamate were studied over a period of > 5 h (Fig. 2). Chloroplasts, after treatment with the herbicide, were allowed to stand in the apparatus for 5 min before the experiment started. The results showed that proton translocation noticeably decreased with time at different concentrations of the carbamate. At a higher concentration of PC, chloroplasts showed a lower activity in proton translocation. Kinetic analysis of proton translocation in illuminated chloroplasts Equation (1) was used to plot the graph in Figure 3. A linear graph with a negative slope of K, is expected. The rate of proton uptake through illuminated chloroplast membranes decreased linearly with time (Fig.3). The rate of proton movement is significantly reduced at higher concentrations of the carbamate. Hence, the coefficient of linearity of graph is significantly reduced as the carbamate concentration increases. A typical ph changes in chloroplasts is shown in Figure 4. 97

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8 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL off 6.5 ph a d oat ~ e b Jo Time (sec) b c Figure 4 Light-induced ph changes in thylakoids from spinach chloroplasts treated with dithiocarbamate at (a).2 ram, (b) 3 ~tm, (c) 3 nm, (d) 25 ram, (e) control. Light is turned on and off at indicated times. Kinetic analysis of proton transtocation through chloroplast membranes after illumination Likewise, equation (2) was used to study the kinetics of proton translocation in the dark (Figure 5). The rate of proton leakage decreases linearly at low carbamate concentration but significantly deviates from linearity as the carbamate concentration increases. The results also show that the rate of proton uptake is faster than that of proton leakage. Change of rate constants of proton uptake and leakage Both rates of proton uptake and leakage (KL, KD) through chloroplast thylakoids change noticeably over a long period of study ( > 5 h ) at various concentrations of the carbamate. The variation of the proton uptake (KL) and the proton leakage (KD) constants with the concentration of the carbamate are shown, in Figure 6. KL increases slowly with the concentration of PC while Kt, varies nonlinearly with the increase of PC concentration. The initial rate (R) of proton translocation was also 974

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11 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL calculated at various concentrations of PC. The results are shown in Table. The rate decreases with the PC concentration. Discussion Pyrrolidine carbamate is an effective insecticide but its effect on plants remains sketchy. Pesticides can cause membrane disruption by inducing lipid peroxidation and disrupt other enzymatic functions in the Calvin cycle and biosynthetic pathways [9-12]. They inhibit electron flow in the photosystems and cause accumulation of the chlorophyll precursor, protoporphyrin X [9-1]. Specific examples include triazines, ureas and uracils which are known to inhibit electron flow in photosystem during the light reaction of photosynthesis [11 12]. The pigments, proteins and enzymes involved in the photosynthesis reactions are located in chloroplasts. Many pesticides are also known to inhibit photosynthesis reaction complexes by binding to the quinone-binding protein, D1 protein, which results in stopping electron flow through photosystem [13]. The D1 protein, formerly known as the 32-kDa or QB protein, consists of 353 amino acid residues and constitutes five transmembrane segments. The D1 Table nitial rate of proton translocation Concentration of Pyrrolidine dithiocarbamate o ( o.trol) lonm 3rim 66 nm 1~r 3gM KL (s" b ~ss (nmole H*/mg Chl) nitial rate of proton pumping (R) (mole H+/mg Chl. per sec) ~ ~am raM

12 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL protein is rapidly degraded and synthesized during normal photosynthetic functions [14-15]. n this study, PC was used to study its effects on proton transport through spinach chloroplasts. The herbicide inhibits the proton gradient formation by CF.CF~ complexes in spinach chloroplasts by inhibiting proton translocation. PC produces a significant inhibition of the proton transport across chloroplast membranes. This would subsequently affect the ATPase function via uncoupling reactions or interfere with electron transport and energy transfer in chloroplasts. The carbamate is believed to act on membranes in which phosphorylation takes place at a similar site. PC apparently has high affinity noncovalent binding at the site. The kinetic analysis of proton translocation with PC show the first order of rate of proton transport at a low carbamate concentration (Fig. 3). However, at a relative higher concentration of PC (> 66 gm), the first order kinetic in proton transport diminishes. The coefficient of linearity decreases and the typical profile of ph changes becomes distorted (Fig. 4). t is believed that the tertiary conformation of the membrane for proton translocation is significantly altered. When the tertiary structure is not significantly disrupted, proton translocation is observed but markedly reduced. Likewise, the kinetic of proton translocation in the presence of a trace amount of the carbamate was studied after the light was turned off (Fig. 5). Similar trends were observed as those under illumination. The preparation of chloroplast membranes from fresh spinach was good for a week or two. Both the rate constants of proton translocation are dependent on the duration of the experiment and the concentration of PC. The PC reaction possibly caused significant disruption on the proton channel; thus, both KL and KD appeared to increase sligt)tly with time. The kinetic data on proton movement show the first order of rate of translocation with PC at a low concentration while inhibitory effects become significant at a higher concentration. Accumulated protons 978

13 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL probably leak back out from the carbamate-treated chloroplasts through a different pathway in the dark. Hence, it took a longer time. Disruption of membrane integrity is believed to cause the different kinetics. n conclusion, PC inhibits proton translocation through chloroplast membranes. t may also act at a few target sites on the chloroplast membranes. Even though the carbamate does not significantly damage crops and higher plants, there may be insidious effects as a result of interruption of proton translocation at the site of action. The inhibitory effects would disturb the ATPase activity. Thiocarbamate is known to inhibit lipid synthesis as one of the modes of action. However, the mechanism of inhibition is unknown. A mechanistic understanding of its effects is useful in elucidating the interaction between pesticides and vital enzymatic activities. Acknowledgement This work was supported in part by a research grant (no.371/95m) from RGC. ESMP also acknowledges the graduate assistantship from CUHK. Part of the results were presented at the Asia-Pacific Conference on Science and Coastal Environment in June (HK). References Ho,Y.K. and Wang,J.H. (1981) J. Bioenerg. Biomembr. 13(5), Lein, S., Racker, E. (1971) Methods Enzymol. 23, Reimer, S., Selman, B.R. (1981) J. Biol. Chem. 253, Anthon, G.E. and Jagendorf, A.T. (1983) Biochim. Biophys. Acta 723, Lynn, W.W. (1968) Biochemistry, 7, Ho, Y.K. and Wang, J.H. (1981) J. Biol. Chem. 256, Portis Jr.,A.R. and McCarty, R.E. (1976) J. Biol. Chem. 251, Arnon, D. (1949) Plant Physiol. 24, Fuerst, E.P.and Norman, M.A. (1991) Weed Sci. 39, Smith, A.G., Marsch, O., Elder, G.H. (1993) Biochem. J. 292, Dilley, R.A. and Schreiber, U. (1984) J. Bioenerg. Biomembr. 16, Steinrucken, H.C. and Amrhein, N. (198) Biochem Biophys. Res. Commun. 94,

14 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL 13. Kyle, D.J. (1985) Photochem. Photobiol, 41, Kyle, D.J., Ohad,. and Arutzen, C.J. (1984) Proc. NatL. Acad. Sci. U.S.A. 81, Prochaska, L.J.and Dilley, R.A. (1978) Biochem. Biophys. Res. Commun. 83,