Cd2+ Ions Affect the Quaternary Structure of Ribulose-1, 5-bisphosphate Carboxylase from Barley Leaves

Biochem. Physiol. Pflanzen 183, 371-378 (1988) VEB Gustav Fischer Verlag Jena

Cd2+ Ions Affect the Quaternary Structure of Ribulose-l ,5-bisphosphate Carboxylase from Barley Leaves MARIE STIBOROV A Department of Biochemistry, Faculty of Natural Sciences, Charles University, Prague, Czechoslovakia Key Term Index: cadmium, inhibition, quaternary structure, ribulose-I,5-bisphosphate carboxylase; Hordeum vulgare

Summary The Cd2 + ions inhibit ribulose-I ,5-biphosphate carboxylase (RuBPC) isolated from barley (Hordeum vulgare L.) leaves. The inhibition is irreversible. This inhibition can be ascribed to the formation of mercaptides with the enzyme's thiol groups. The effects of the Cd 2 + ions on the changes of the quaternary structure of the RuBPC protein molecule are examined. The modification ofthiol groups by Cd2+ ions results in the dissociation oflarge and small subunits of the enzyme. The dissociation is irreversible. The role ofthe Cd 2 + -induced inhibition of RuB PC in the mechanism of aCd2 + toxicity on photosynthesis is discussed.

Introduction Cadmium is one of the most toxic heavy metals. Photosynthetic processes of higher plants are particularly susceptible to Cd2+ (BASZYNSKI et al. 1980; BASZYNSKI 1986; CLUSTERS and VAN ASSCHE 1985; JASTROW and KOEPPE 1980). Cd 2 + has been found to reduce the chlorophyll content (BAZZAZ etal. 1974; HAMPpetal. 1978; STIBOROV Aet al. 1986a; SToBARTet al. 1985) and oxygen evolving reactions of photosystem II (BASZYNSKI et al. 1980; BAZZAZ and GOVINDJEE 1974). Recently, BASZYNSKI (1986) described that reduced photosystem II activity and reduced non-cyclic photophosphorylation rates in chloroplasts of Cd 2 + treated plants is accompanied by decrease of the photosynthetic NADPH and ATP production and these facts may be the cause of Cd 2 + -induced photosynthetic COrfixation inhibition. However, he concluded that the involvement of dark reactions in the inhibitory effect of Cd 2 + cannot be excluded (BASZYNSKI 1986). Indeed, WEIGEL (1985) reported that Cd2+ affects photosynthesis by inhibition of different reaction steps of the Cal vin cycle. Cd 2 + -induced alteration of ribulose1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) activity may be one of the possible reasons for inhibition (WEIGEL 1985). Indeed, the activity of RuB PC was inhibited by Cd 2 + ions in vivo and in vitro (ERNST 1980; STIBOROVA et al. 1986a, b). The present study continues our studies of the effects of heavy metals on photosynthesis (STIBOROVA et al. 1986a, b, c) and is aimed at finding the mechanisms of inhibitory effects of Cd2 + on RuBPC at the molecular level. The molecular structure of RuB PC is known. The enzyme from higher plants is comprised of 8 large (L; ~55,000 daltons) and 8 small subunits (S; ~ 14,000 daltons), with the hexadecameric holoenzyme designated as L 8 S8 . The functions of both subunits are, however, not quite clear though they have been intensively studied. The lattest results indicate that both BPP 183 (1988) 5

371

subunit types are required for catalysis by RuBPC and the L 8 Sg structure of the enzyme represents the catalytic entity (JORDAN and CHOLLET 1985; McFADDEN et al. 1986). It was found earlier that modification of SH groups of the RuBPC protein molecule can destabilize the quaternary structure of the enzyme and this modification can result in enzyme dissociation into subunits (NISHIMURA eta!' 1973; TAKABE and AKAZAWA 1975 a, b). As Cd2+ ions are known as thiol modifying agents, their effects on the quaternary structure of the RuBPC protein melecule are studied in the present paper.

Material and Methods Plant material and chemicals used Barley plants (Hordeum vulgare L. cv. Spartan) were cultivated at 20 to 30°C in a greenhouse under natural light. 2-Mercaptoethanol and dithiothreitol from Koch-Light Laboratories, Ltd. , Colbrook, England , RuBP by Serva GmbH , Heidelberg, FRG , Sepharose 4B by Pharmacia Fine Chemicals, Uppsala , Sweden , and other chemicals from Lachema Brno, Czechoslovakia, all of analytical grade purity, were used in the experiments. Na214C03 (1 ,957 MBq mmoll - 1) was product ofthe In stitute for Research, Production and Uses of Radioisotopes (Prague, Czechoslovakia). Determination of RuBPC activity RuBPC activity was measured b y adding 0.1 ml aliquots of sample to 0.4 ml of acti vation medium containing 100 mMTris-HClbufferpH 8.0,OA mMEOTA , 10 mMMgCh , 1 mMdithiothreitol and5 mMNa214C03at20 °C. Following 15 min of activation, reactions were initiated by adding 50 III 10 mM RuBP. Reactions were stopped after 5 min by add ing 0.5 ml of 6 M H CI. The RuBPC activity was measured as 14C fixed into acid-stable reation product in a toluene scintillation cocktail on aPackard TRI CARB 300 liquid sci ntill ation counter. The activity was measured at 37°C. Isolation of RuBPC RuBPC was isolated by the modified procedure of RYAN and TOLBERT (1975). The enzyme was extracted from 3 week old pl ants of barley and precipitated by ammoni um sulphate (40- 60 %) , chromatographed on a OEAE-cellulose column and on a Sepharose 4B column. The detailed isolation process is described earlier (OOUBRAVOVA 1985). Inactivation of RuBPC by Cd2 + ion;The inactivation measurements were carried out in 3 ml test tubes using a total volume of I ml incubati on mixture in 0.1 M Tris-HCl buffer pH 7.2 with the suitable Cd2+ (3 CdS04 . 8 H2 0) concentrations. After a suitable incubation period an O. I ml aliquot of the mixture was transferred into the reaction medium for the enzyme activity determination and the activity was measured (see above). Controls in the absence ofCd 2 + were carried out in parallel. The incubation time was 0- 50 min. The concentration of RuBPC was 111M. The inactivation was performed at 20 °C. The samples of RuB PC (10 11M) incubated with 1, 5 and 10 11M Cd H for 5 hin 50 mM Tris-HCI buffer p H 7.2 at 20°C were chromatographed on a Sepharose 4 B column (2 x 50 cm) and the changes in the quaternary structure of the enzyme were investigated. Elution was carried out by the buffers in whi ch the enzyme was incubated. A Sepharose 4 B column (2 x 50 cm) was calibrated with the following standard proteins: thyroglobulin (669,000), urease (483,000), maize phosphoenolpyruvate carboxylase (400,000), catalase (232,000), lactate dehydrogenase (140,000), and bovine serum albumin (67 ,000). Sodium dodecylsulfate (SDS) electrophoresis Vertical 15 % polyacrylamide slab gels in 0.1 M Tris-HClpH 8.0 and 0.2 % SOS were used. The same buffer was used for upper and lower reservoirs. Proteins (native RuBPC and protein peaks from a Sepharose 4B column chromatography) were prepared for electrophoresis by heating in 0.1 M Tris-HC1 , pH 8. 0, 1 % SOS , 0 . 1 M 2mercaptoethanol and a trace of bromphenol blue fo r 5 min at 100 °C. SOS-electrophoresis was performed at 20 rnA until the tracki ng dye had mi grated completely off the gel. The protein bands were visualized by staining with Coomassie brilliant blue R-250 . Bovine serum albumin (67,000), ovalbumin (43,000) and lysozyme (14,000) were used as standards.

372

BPP 183 (1988) 5

Results The activity of barley RuBPC was inhibited by Cd2+ ions when the isolated enzyme was preincubated with this metal ion (Fig. 1, Table 1). The Cd2 + ions caused a time-dependent loss of activity of RuBPC (Fig. 1). Table I. Rate constants of RuBPC inactivation by Cd2 + ions. Experimental conditions are described in the text (see Material and Methods - Inactivation of RuBPC by Cd2 + ions). The rate constants of inactivation were calculated from pseudo-first order reactions. Rate constant of inactivation (Sl)

1.28 . 3.13· 7.18. 15.27 .

1.0 2.5 5.0 50.0

10- 4 10- 4 10- 4 10- 4

The reaction of Cd 2 + ions with the enzyme followed pseudo-first-order kinetics. This can be concluded from the linear nature of the relationships shown Fig. 1 where activity is plotted against time in a semi-logarithmic plot. The rate of inactivation of RuB PC by Cd 2 + ions was proportional to reagent concentrations (results not shown). In order to determine whether the 0.75

.-----.

.., 0.50

,..100 ~ 50 .t 25 \01 10

52

..

-

...

-;- 0.25

"

~

;;;-

~

0

til)

.2

1 0

10

20

30

time (min)

1

40

50

o

0.25

0.50 0.75 I/ Cd 2+(pM- 1) 2

Fig. 1. Semi-logarithmic plots of RuBPC inactivation versus time with different Cd 2 + concentrations. Assay conditions: 0.1 M Tris-HCI buffer pH 7.2. a-I [!M Cd 2 + ; b - 5 [!M Cd 2 + ; C - 50 [!M Cd 2 + Fig. 2. Double-reciprocal plot of inactivation rate constants versus Cd 2 + concentrations. Assay conditions as in Fig. 1.

inactivation of RuBPC by Cd 2 + ions occurs through the formation of a noncovalent complex between the enzyme and the inhibitor prior to the covalent modification reaction the following calculation was done. The y-intercept of a double- reciprocal plot of the observed pseudo-firstorder rate constants (Table 1) versus Cd 2 + concentrations gave a value close to zero (Fig. 2). This result implies that a reversible noncovalent enzyme-inhibitor complex is not formed (LEVY et al. 1963). Thus, the inactivation seems to occur through a simple bimolecular mechanism. The reaction order with respect to Cd 2 + ions was 1.0. It is given by the slope of the BPP 183 (1988) 5

373

straight part of the logarithmic plot of the pseudo-first-rate constants (k) versus the logarithmic plot of Cd2+ concentration (not shown). Cd 2 + ions are known as thiol inhibitor (AYLETT 1978; WEBB 1966). The reversibility of the inhibitory effect of Cd2+ ions by a reductant of disulfide bonds such dithiothreitol provides information about the mechanism of action of this inhibitor. Cd 2 + -induced inactivation of RuB PC is stopped by the addition of 1 mM EDTA (Fig. 3). However, the subsequent addition

-0

0-

>-

75

~

';

'+:\I

to

l

i 0

20

2

t

50

70

100

time (min) Fig. 3. Effect oj EDTA and dithiothreitol on the inactivation ojRuBPC by 501lM Cd 2 + The arrows I and 2 indicate the sequential addition of I mM EDTA and IOmM dithiothreitol, respectively. Assay conditions as in Fig. I.

of 10 mM dithiothreitol into the incubation mixture did not influence the inactivation. No reversal of inactivation was observed (Fig. 3). The lack of reversal by dithiothreitol on the inactivation of RuBPC by Cd 2 + ions suggests the formation of mercaptides. The effect of Cd 2 + ions on the quaternary structure of the enzyme protein molecule was studied by the chromatography on a Sepharose 4 B column. The incubation of the native enzyme protein with Cd 2 + ions resulted in conversion of the quaternary structure of RuBPC. The dissociation of the enzyme protein molecule was observed (Fig. 4). When RuBPC (10 [lM) was incubated with 5-10 [lM Cd 2 + ions, 2 well-resolved peaks of proteins were separated by a Sepharose 4B column chromatography (Fig. 4). Cd 2 + ions at concentrations of 1 [lM caused incomplete dissociation (Fig. 4). Calibration of the Sepharose 4B column with standard proteins and analysis of the peaks A280 nm fractions after Cd 2 + treatment revealed that the first protein peak likely contained octamers of L subunits of RuB PC (molecular mass of the first peak was about 440,000), while the second protein peak has molecular mass of about 110,000 (Fig. 5). Indeed, the analysis of the first protein peak by sodium dodecylsulfate (SDS) electrophoresis on slab gels indicated that first peak contained only L subunit (Fig. 6). No S subunit was found. The SDS electrophoresis analysis of second protein peak was not carried out, because the protein concentration of this fraction was too dilute to permit analysis by SDS electrophoresis. No detectable RuBPC activity of both protein fractions was found (Fig. 4). Furthermore, L fraction and further protein fraction were brought to I mM EDTA and 10 mM dithiothreitol by the addition of EDT A and dithiothreitol and dialyzed against 50 mM Tris-HCI buffer pH 7.0 containing I mM EDTA and 10 mM dithiothreitol overnight (at 4°C) to remove excess of Cd2+ ions. No detectable RuBPC activity was found for both protein fractions after dithiothreitol treatment. Moreover, L subunit fraction obtained after EDT A and dithiothreitol pretreatment was mixed with protein fraction with molecular mass of 110,000 and after 10 min incubation (20°C) the RuBPC activity was measured. No enzyme activity was detected after 374

BPP 183 (\988) 5

0.4

A

0.2

1

04 .

0.2

0

0

E 0.2 c

B

0.4

0

~ 0.1

0.2.:r

~o

"0 E 0.4.3-

i

0

0.2 C

ofo 0.1

0.2

.c '"

teo

'"

....,..

'>

:s te

0 u 0.4 ~

0.2 D

:I

III:

0.1

0

0.2 40

80

120

0

elution volume (ml)

Fig. 4. Chromatography of RuBPC without (AJ and with I flM Cd 2 + (B), 5 flM Cd 2 + (C) and IOflM Cd2+ (D) on Sepharose 4B. Assay conditions: RuBPC (I 0 !!M) was preincubated in 50mM Tris-HCI buffer pH 7.2 (5h) without (A) and with Cd 2 + (B -O) and applied on a Sepharose4B column (2 x 50cm). The elution was carried out by buffers in which the enzyme was preincubated. Arrows I , 2 and 3 indicate location of LgSs, Ls and the protein fraction with molecular mass of about 110,000, respectively. Proteins ( )and the RuBPC activ ity (0 , .. .. ·0).

these reconstitution experiments, neither. When the mixture obtained after the reconstitution experiments was chromatographed on a Sepharose 4B column, 2 protein peaks were obtained again (not shown). Thus the dissociation of RuBPC protein molecule which followed from the modification of the enzyme thiols by Cd 2 + was irreversible.

Discu ssion RuBPC comprises the largest fraction of the soluble protein of leaves (JENSEN and BAHR 1977) and its activity is considered to be the prime determinant of C 3 -leaf photosynthesis rate at low internal leaf CO 2 concentrations (FARQUHAR 1982). As thi s enzyme plays a key role in photosynthesis , the effect of Cd 2 + ions, which are known as toxic metal ions for photosynthesis, is studied in the present paper. The in vitro experiments presented in this paper indicate that this enzyme is sensitive to Cd 2 +. The Cd2+ ions may inhibit the RuBPC by formation of mercaptides with thiol groups of cysteine, which are present in the protein (LORIMER 1981). The irreversible modification of thiol groups by Cd2 + ions correlates with the changes of the quaternary structure of the enzyme molecule . The dissociation of LgSs native molecule to large and small subunits was observed. No reversal of Cd 2 + -induced inhibition and dissociation was obtained . When the same concentration of CdS0 4 and RuBPC were used in the experiments, 50 % inhibition of the enzyme activity was observed after 50 min incubation (1 IlM Cd 2 + and 1 IlM BPP 183 (198 8) 5

375

i

i~

first I native RuBPC 4OL-__L -____L - - i_ _L-LJ~

I

2 3 4 5 6 molecular mass JC.IO 5

Fig. S. Th e molecular masses determinacion. Assay conditions: standard proteins were dissolved in SOmM Tris-HCl buffer pH 7.2 (Smgml' l ) and applied on a Sepharose 4B column (2 x SOcm). The elution volumes of standards were plotted versus log of their molecular masses. BSA - bovine serum albumin , LDH - lactate dehydrogenase, PEPC - phosphoenolpyruvate carboxylase, TH - thyroglobulin .

Fig. 6. SDS-polyacrylamide gel electrophoresis of standard proteins (I). native RuBPC (2), and the first protein peak obtained from Sepharose 4B after Cd 2+ treatment (3). Assay conditions are described in the text (see Material and Methods). a - bovine serum albumin , b - ovalbumin, c - lysozyme, L - large subunit and S - small subunit of RuBPC.

376

BPP 183 (1988) 5

RuBPC - Fig. 1). However, already 1 f.tM and 5 f.tM concentrations of Cd 2 + partially or totally disrupt the native RuB PC molecule (10 f.tM) into Lg fraction and further protein fraction with molecular mass of 110,000 after 5 h incubation, respectively. Thus, for the prolonged effect of Cd2+ on RuBPC, the lower concentrations of metal ions with respect to the enzyme concentrations produce the inactivation. It was estimated that the concentration of RuBPC in chloroplasts is about 0.4-0.5 mM (JENSEN and BAHR 1977). Thus, it can be supposed from the above mentioned results that the Cd 2 + concentrations in chloroplasts of about 50-250 f.tM could produce the alteration of RuBPC protein molecule to inactive forms in vivo. It was found in our earlier paper in the in vivo experiments that 50 and 100 % inhibition of RuBPC produced by about 11 f.tM and 60 f.tM intemalleaf Cd2+ concentrations, respectively, (STIBOROV A et al. 1986 a). Hence, the results obtained from the in vivo experiments correlate with the in vitro results presented in this paper. The differences between in vivo vs. in vitro results, respectively, could be caused by different microenvironmental conditions (pH, the presence of substrates and effectors, etc.). Furthermore, the discrepancy can also follow from the fact that the actual concentrations of Cd2+ in chloroplasts are not known. In our previous studies these concentrations were not measured. Only the total leaf concentrations of Cd 2 + have been detected (STIBOROVA et al. 1986a). Although it is known that Cd 2 + tends to accumulate in the chloroplasts (ERNST 1980), actual information on the Cd 2 + concentration in this organelle in vivo is very scarce (CLIJSTERS and VAN ASSCHE 1985). It can be concluded from present results that RuB PC can be a target for the toxic action of Cd 2 + on photosynthesis, because the other photosynthetic processes are influenced only by higher Cd2+ concentrations. Only the photosynthetic photophosphorylation is an exception. Both cyclic and non-cyclic photophosphorylation are inhibited by 5 f.tm concentrations of Cd2 + ions (LUCERO et al. 1976). On the other hand, biosynthesis of chlorophylls is strongly inhibited (98 %) by Cd 2 + ions only at concentrations of about 10- 2 M (STOBART et al. 1985). Furthermore, 0.2 and 0.5 mM concentrations of Cd(N03h produced 50 % inhibition of photosystem II reactions in chloroplasts from maize and spinach, respectively (BAZZAZ and GOVINDJEE 1974; LI and MILES 1975). For better understanding of the mechanisms of Cd 2 + action on photosynthesis at the physiological and biochemical level, further studies, which use the combination of the in vivo and the in vitro experiments with the investigations of all photosynthetic processes in these experiments must be carried out.

References AYLETT, B. J.: The chemistry and bioinorganic chemistry of cadmium. In: WEBB, M. (Ed.): The chemistry, biochemistry and biology of cadmium, pp. 1-43. Elsevier/North-Holland Biomedical Press, Amsterdam 1978. BASZYNSKI, T.: Interference of Cd2 + in functioning of the photosynthetic apparatus of higher plants. Acta Soc. Bot. Pol. 55, 291- 304 (1986). BASZYNSKI, T., WAJDA, L., KR6L, M., WOLINSKA, D., KRUPA, Z., and TUKENDORF, A.: Photosynthetic activities of cadmium-treated tomato plants. Physiol. Plant. 48, 365-370 (1980). BAZZAZ, J, E" and GOVINDJEE: Effects of cadmium nitrate on spectral characteristics and light reactions of chloroplasts. Environ. Lett, 6, )-12 (1974), BAZZAZ, p, A., ROLFE, G. L., and CARLSON, R. W,: Effect of cadmium on photosynthesis and transpiration of excised leaves of corn and sunflower. Physiol. Plant. 32, 373-376 (1974),

25

BPP 183 (1988) 5

377

CWSTERS, H., and VAN ASSCHE, F.: Inhibition of photosynthesis by heavy metals. Photosynth. Res. 7, 31-40 (1985). DOUBRAVOVA, M.: Thesis. Faculty of Natural Sciences. Charles University, Prague 1985. ERNST, W. H. 0.: Biochemical aspects of cadmium in plants. In: NRIAGU, J. O. (Ed.): Cadmium in the Environment., Part I, pp. 630-653. Wiley Interscience, New York-Chichester 1980. FARQUHAR, G. D.: Stomatal conductance and photosynthesis. Ann. Rev. Plant Physiol. 33, 317-345 (1982). HAMPP, R., BEULICH, K., and ZIEGLER, H.: Effects of zinc and cadmium on photosynthetic COrfixation and the Hill activity of isolated spinach chloroplasts. Z. Pflanzenphysiol. 77, 336- 344 (1976). JASTROW, J. D., and KOEPPE, D. E.: Uptake and effects of cadmium in higher plants. In: Cadmium in the Environment. Part I, pp. 608-637. Wiley Interscience, New York-Chichester 1980. JENSEN, R. G., and BAHR, J. T.: Ribulose 1,5-biphosphate carboxylase/oxygenase. Ann. Rev. Plant Physiol. 28, 379-400 (1977). JORDAN, D. B., and CHOLLET, R.: Subunit dissociation and reconstitution of ribulose-l ,5-bisphosphate carboxylase from Chromatium vinosum. Arch. Biochem. Biophys. 236,487-496 (1985). LEVY, H., LEBER, P., and RYAN, E.: Inactivation of myosin by 2-4 dinitrophenol and protection by adenosine triphosphate and other phosphate compounds. J. BioI. Chern. 238, 3654-3659 (1963). LI, E. H., and MILES, C. D.: Effects of cadmium on photoreaction II of chloroplasts. Plant Sci. Lett. 5,33-40 (1975). LORIMER, G. M.: The carboxylation and oxygenation of ribulose-l,5-bisphosphate: the primary events in photosynthesis and photorespiration. Ann. Rev. Plant Physiol. 38, 349-383 (1981). LUCERO, H. A., ANDREO, C. S., and VALLEJOS, R. M.: Sulfhydryl groups in photosynthetic energy conservation. III. Inhibition of photophosphorylation in spinach chloroplasts by CdCLz. Plant Sci. Lett. 6, 309-313 (1976). McFADDEN, B. A., TORRES-RVIZ, J., DANIELL, H., and SAROJINI, G.: Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota. Phil. Trans. R. Soc. Lond. 8313, 347-358 (1986). NISHIMURA, M., TAKABE, T., SUGIYAMA, T., and AKAZAWA, T.: Structure and function of chloroplast protein: XIX. Dissociation of spinach leaf ribulose-l,5-bisphosphate carboxylase by p-mercuribenzoate. J. Biochem. 74, 945-954 (1973). RYAN, F. J., and TOLBERT, N. A.: Ribulose diphosphate carboxylase/oxygenase. 3. Isolation and properties. J. BioI. Chern. 250,4229-4233 (1975). STIBOROVA, M., DOUBRAVOVA, M., BREZINOVA, A., and FRIEDRICH, A.: Effect of heavy metal ions on growth and biochemical characteristics of photosynthesis of barley (Hordeum vulgare L.). Photosynthetica 20, 418-425 (1986a). STIBOROVA, M., DouBRAvovA, M., and LEBLOVA, S.: A comparative study of the effect of heavy metal ions on ribulose-l ,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase. Biochem. Physiol. Pflanzen 181, 373-379 (1986b). STIBOROV A, M. , HROMADKOV A, R. , and LEBLOV A, S.: Effect of ions of heavy metals on photosynthetic characteristics of maize (Zea mays L.). Biologia 41, 1221-1228 (1986c). STOBART, A. K., GRIFFITHS, W. T., AMEEN-BuKHARI, I., and SHERWOOD, R. P.: The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley. Physiol. Plant. 63, 293-298 (1985). TAKABE, T., and AKAZAWA, T.: Further studies on the subunit structure of Chromatium ribulose-l ,5-bisphosphate carboxylase. Biochemistry 14, 46-50 (l975a). TAKABE, T., and AKAZAWA, T.: The role of sulfhydryl groups in the ribulose-l,5-bisphosphate carboxylase and oxygenase reactions. Arch. Biochem. Biophys. 169,686-694 (l975b). WEBB, J. J.: Sulfhydryl reagents. In: WEBB, J. J. (Ed.): Enzyme and Metabolic Inhibitors, Vol. II, pp. 635-653. Academic Press, New York 1966. WEIGEL, H. J.: Inhibition of photosynthetic reactions of isolated intact chloroplasts by cadmium. J. Plant Physiol. 119, 179-189 (1985).

Received April 20, 1987; revised form accepted July 29, 1987 Author's address: Dr. MARIE STIBOROVA, Department of Biochemistry, Faculty of Natural Sciences, Charles University, Albertov 2030, 12840 Prague 2, Czechoslovakia.

378

BPP 183 (1988) 5