I. Plastocyanin Content and Reactivity

932 P. V. SANE AND G. A. HAUSKA

The activation of the NADP-specific glyceralde- enzyme complex should be widely distributed in the hyde phosphate dehydrogenase in vivo was demon- plant kingdom. strated in variety of plants 17, 27. If this effect is re- This study was made possible by a grant and finan- garded as pars pro toto, the labile C02-fixing cial support from the Deutsche Forschungsgemeinschaft.

1 J. A. BASSHAM, Advances Enzymol. 25, 39 [1963]. 15 T.BÜCHER, in: Meth.Enzymol., Vol.1, 415, ed. S.P.COLO- 2 J. A. BASSHAM, Annu. Rev. Plant Physiol. 15, 101 [1964]. WICK and N. O. KAPLAN, Academic Press, New York 1955. 3 R. S. CRIDDLE, in Biochem. Chloroplasts, Vol. I, 203, ed. 16 B. MÜLLER, Biochim. biophysica Acta [Amsterdam] 205; T. W. GOODWIN, Academic Press, New York 1966. 102 [1970]. 4 A. C. RUTNER, Biochemistry 9, 178 [1970]. 17 H. ZIEGLER and I. ZIEGLER, Planta 65, 369 [1965]. 5 G. VAN NOORT, W. HUDSON, and S. G. WILDMAN, Plant 18 H. ZIEGLER, I. ZIEGLER, and H.-J. SCHMIDT-CLAUSEN, Physiol. 36, Suppl. XIX [1961]. Planta 67,344 [1965]. 6 L. MENDIOLA and T. AKAZAWA, Biochemistry 3, 174 19 E. LATZKO and M. GIBBS, in Progr. Photosynth. Res., Vol. [1964], ILL, 1624, ed. H. METZNER, 1969. 7 G. VAN NOORT and S. G. WILDMAN, Biochim. biophysica 20 B. MÜLLER, I. ZIEGLER, and H. ZIEGLER, Europ. J. Bio- Acta [Amsterdam] 90, 309 [1964]. chem. 9,101 [1969]. 8 P. W. TROWN, Biochemistry 4, 908 [1965]. 21 I. ZIEGLER, II. Intern. Congr. Photosynth. Res., Stresa. 9 J. M. PAULSEN and M. D. LANE, Biochemistry 5, 2350 Italy 1971. [1966]. 22 W. HOOD and N. G. CARR, Planta 86, 250 [1969]. 10 B. MÜLLER and H. ZIEGLER, Planta 85, 96 [1969]. 23 R. B. PARK and N. G. PON, J. molecular Biol. 3, 1 [1961]. 11 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR, and R. J. 24 M. D. SCHULMANN and M. GIBBS, Plant Physiol. 43, 1805 RANDALL, J. biol. Chemistry 193, 265 [1951]. [1968], 12 D. I. ARNON, Plant Physiol. 24,1 [1949]. 25 J. F. REITHEL, Adv. Prot. Chem. 18, 123 [1963]. 13 J. HURWITZ, A. WEISSBACH, B. L. HORECKER, and P. Z. 26 L. J. REED and D. J. Cox, Annu. Rev. Biochem. 35, 57 SMYRNIOTIS, J. biol. Chemistry 218, 769 [1956]. [1966]. 14 U. HEBER, N. G. PON, and M. HEBER, Plant Physiol. 38, 27 E. STEIGER, I. ZIEGLER, and H. ZIEGLER, Planta 96, 109 355 [1963]. [1971].

The Distribution of Photosynthetic Reactions in the Chloroplast Lamellar System I. Plastocyanin Content and Reactivity

P. V. SANE * and G. A. HAUSKA Abteilung für Biologie, Ruhr-Universität Bochum

(Z. Naturforsch. 27 b, 932—938 [1972] ; received May 15, 1972)

Plastocyanin, Chloroplast fragmentation, Photosystem I, Stroma Lamellae, pH-optimum for electron transport Plastocyanin is released from chloroplasts by sonication, by passing through the French-press, by extraction with detergents, by treatment with organic solvents and by freezing and thawing. No plastocyanin is liberated during osmotic shock of either class I or class II chloroplasts, or by extraction of class II chloroplasts with dilute solutions of EDTA. Under special conditions it is possible to separate grana and stroma lamellae, which retain plastocyanin. The distribution of plastocyanin between these membrane fractions parallels the distribution of photosystem I. The different reactivity of plastocyanin with various chloroplast membrane preparations is not always correlated to plastocyanin content. Other parameters, like pH, seem to influence the interaction of plastocyanin with the surface of the chloroplast membrane.

The function of a biological membrane is related cussed in a recent review 1. With the develpoment of to its molecular architecture (membrane topography) methods to separate distinct regions of the chloro- as well as to its higher organization (membrane plast lamellar system2' 3 it is feasible to study the morphology). The possible relation of structure and distribution of activities and components among function in the chloroplast membrane has been dis- them. Plastocyanin, an electron transport component

Requests for reprints should be sent to Dr. G. HAUSKA, ** Abbreviations used: Tricine for (A-(Tris-hydroxymethyl) - Lehrstuhl für Biochemie der Pflanzen, Abteilung Biologie, methyl) -glycin, MES for 2 (A-morpholino) ethane sulfonic D-4630 Bochum-Querenburg, Postfach 2148. acid, HEPES for A-2-hydroxyethylpiperazine-A-2-ethane * Present adress: Shraddhanand Peth, Post Ajami, Nagpur/ sulfonic acid. India. PLASTOCYANIN IN FRAGMENTED CHLOROPLASTS 933

functioning close to photosystem I, is a good can- of NADPH was measured at 340 nm in a Zeiss spectro- didate for such an enterprise, and this paper is con- photometer, Model PMQ2, modified for illumination from the side. A yellow glass filter (Schott GG 475) cerned with it. We will show that this copper pro- and a heat filter was put into the actinic light path. A tein follows the distribution of photosystem I acti- second monochromator was mounted in front of the vity in the lamellar system. After we had started our phototube. The light intensity of the actinic beam was work a report with similar concern appeared 4. 2.8 x 105 ergs/cm2 per sec measured with a YSI-Ket- In addition we will give further evidence for the tering radiometer. NADP reduction for measurement of plastocyanin was superior to the previously employed necessity to distinguish between the original func- oxygen uptake via methyl viologen, because the sensi- tion of plastocyanin in the chloroplast membrane tivity of the spectrophotometer exceeded that of the and secondary effects exerted by readdition of this oxygen electrode, though the absolute rates of electron protein to deficient membrane preparations 5. transport were lower. If, however, maximal rates of plastocyanin dependent electron flow through photosystem I were desired, methyl viologen catalyzed oxy- Methods gen uptake was measured (see Table 3) 10. Liberation of plastocyanin by incubation of chloro- Preparations plast preparations in 2% Triton X-100 was carried out as described 5. If sonication was used for the liberation The isolation of broken chloroplasts from field grown we proceeded as follows. A known amount of chloro- spinach, as well as osmotic shock and extraction of plasts (0.2 to 1 mg chlorophyll) in 1 ml of a hypotonic chloroplasts by EDTA were carried out as described 6. medium, containing 20 MM Tricine-NaOH, pH 8, and Class I chloroplasts were prepared according to no more than 0.1 M sucrose, was sonicated for 1 min WALKER 7. in four 15 sec bursts with intermediate cooling on ice, Freezing and thawing of broken chloroplasts was using a Branson 20-K cycle sonifier with the standard carried out at 1 mg chlorophyll per ml in 10 MM NaCl micro tip, 1/s inch diameter, at full output. The resulting in the presence or absence of 0.4 M sucrose. After free- suspension was layered over 35% sucrose and centri- zing at —20° over night the chloroplasts were thawed fuged for 1 hour at 50 thousand rpm in a Beckman at RT. Spinco SW-56 Ti rotor. Aliquots of 0.2 to 0.6 ml from Sonication, in the presence or absence of plasto- the top layer, which were free of chlorophyll, were cyanin, was performed as published recently5. If tested for plastocyanin. Possible inactivation of plasto- plastocyanin was present in the sonication medium the cyanin during Triton X-100 extraction or sonication concentration was 100 pM. was ruled out by passing known amounts of plasto- Fragmentation of chloroplasts by passage through cyanin through the procedures. a French-press and subsequent differential centrifuga- Chlorophyll was measured according to ARNON 1S. tion for the isolation of grana and stroma lamellae has been described previously3. A Sorvall Ribi Cell Fractionator, Model RF-1, at the lowest possible pres- Results sure setting (4000 — 5000 psi) was used throughout the preparations described in this paper. The presence Plastocyanin is known to be liberated from chloro- of 0.4 M sucrose in the chloroplasts suspension proved plasts with the help of organic solvents 14 by soni- to be protective in several ways (see Table 2 of this 15 16 17 and Table 1 of the accompanying paper 8) and did not cation or detergent treatment ' . With the de- affect the fractionation as indicated by the ratios of velopment of a sensitive enzymatic test5'18 it is now chlorophyll a to b. possible to measure resolution of plastocyanin from Digitonin membrane vesicles were prepared ac- chloroplast preparations on a micro scale. Table 1 cording to ANDERSON and BOARDMAN9. An isotonic summarizes our data on the percent liberation of concentration of sucrose during the detergent treatment was beneficial again 10. plastocyanin from spinach chloroplasts by various For heptane treatment of chloroplasts we followed methods. Treatment with Triton X-100 is most ef- the procedure of ELSTNER et al. n. fective. Between 2 and 5 nmoles plastocyanin per mg Plastocyanin, ferredoxin and ferredoxin-NADP re- chlorophyll are extracted (see also Ref. 5). Repeated ductase were purified from spinach as described by extraction does not increase this value, which cor- ANDERSON and MCCARTY 12. responds to one plastocyanin for 220 to 550 chloro- Assays phylls. Liberation of plastocyanin by ultra sound is more convenient since it avoids ammonium sulfate Plastocyanin was measured enzymatically by ascor- precipitation and subsequent dialysis 5. Its efficiency bate-photooxidation as published5, but NADP plus ferredoxin and ferredoxin-NADP reductase replaced depends on the energy of sonication, the sonication methyl viologen as electron acceptor system. Formation time, the geometry of the sonication vessel and to 934 P. V. SANE AND G. A. HAUSKA

Preliminary experiments with subchloroplasts ves- Treatment percent liberated of chloroplasts plastocyanin icles show that these are more resistent to various treatments than broken chloroplasts. Both, Triton Triton X-100 100 X-100 and ultra sound can be used, however, to re- ultra sound up to 100 acetone 90 move plastocyanin quantitatively from subchloro- heptane 80 plast vesicles prepared by French-press or digitonin French-press 60, 80 treatment. digitonin 30, 70 freezing and thawing 30, 70 As further shown in Table 1 treatment with hep- isolation from the leaf 5 tane or acetone and subsequent aqueous extraction osmotic shock < 1 EDTA-extraction < 1 also removed most of the plastocyanin. These two methods have been employed for the large scale pre- Table 1. Liberation of plastocyanin from chloroplasts by paration of plastocyanin n'14. French-press treat- various methods. Chloroplasts were treated by the various ment removes 60 — 80% of the plastocyanin under procedures, and after sedimentation the supernatants were tested for plastocyanin (see Methods). The assay mixture conditions which allow the separation of grana from contained in 1 ml 50 MM Tricine-NaOH **, pH 8.0, 50 MM stroma lamellae 3. In the presence of 0.4 M sucrose NaCl, 5 MM ascorbate, 0.25 MM NADP, 10 /ug ferredoxin, 50 pg ferredoxin-NADP reductase and digitonin-subchloro- less plastocyanin is lost from the chloroplast. Digi- plast vesicles equivalent to 10 jug chlorophyll. If two values tonin does not release all of the plastocyanin, even are given, the lower corresponds to liberation of plastocyanin not at concentrations as high as 1.5%. A high ratio in the presence of 0.4 M sucrose. The amount of plastocyanin released by Triton X-100 was set 100%. of digitonin to chlorophyll and hypotonicity are necessary for maximal liberation. In the presence of 0.4 M sucrose, 1 mg chlorophyll per ml and 0.5% a lower degree, on the osmolarity of the suspending digitonin, only 30 — 50% of plastocyanin are found medium. In a hypotonic medium 30 sec sonication in the supernatant after centrifugation. Freezing and at full output was sufficient to resolve as much thawing of chloroplasts also results in a loss of plastocyanin as by extraction with Triton X-100. plastocyanin from the chloroplasts. Again, an iso- The removal of plastocyanin by sonication is a re- tonic concentration of sucrose is protective, but does versible process 5 which is once more shown in Fig. 1. not fully prevent the effect. Apparently ineffective is If sufficient purified plastocyanin is added before mixing of chloroplasts in a Warring blender during sonication the chloroplasts retain their full comple- isolation from the leaf. Almost no plastocyanin was ment of this copper protein. This should provide the released into the isotonic medium employed by possibility to investigate the effect of ultra sound on 30 sec blending at 0°. No plastocyanin was lost to the chloroplast membrane apart from the loss of the medium by osmotic shock of either class II or plastocyanin. class I chloroplasts. Osmotic shock is known to remove the outer and to modify the inner membrane of the chloroplast. It results in breakage of those parts of the inner membrane which enclose the stroma and causes swelling and unfolding of the residual lamellar system, especially of the grana regions (cf. to Ref. 19). All these structural changes

o a; do obviously not touch the binding of the plasto- Vt o , 3 E 1 cyanin. Moreover, treatment with dilute EDTA-solu- Q- c tions, which is known to remove coupling factor 1 from the membrane 6, did not remove plastocyanin.

sonication time PARK and SANE recently proposed a model for Fig. 1. The liberation of plastocyanin by sonication and its photosynthesis 1 which accommodates both, structure prevention by addition of plastocyanin. Chloroplasts were and function of the chloroplast lamellar system. In sonicated in the presence and absence of 100 /UM plastocyanin 5. After removing excess free plastocyanin by repeated essence we suggested that the grana regions contain centrifugation any plastocyanin bound to the chloroplast photosystem I and photosystem II, while the stroma membrane was liberated by sonication as described under lamellae have photosystem I with possibly a still Methods. The assay mixture for the determination of plastocyanin is given in Table 1. developing, incomplete photosystem II. Thus stroma PLASTOCYANIN IN FRAGMENTED CHLOROPLASTS 935 lamellae and grana are throught in biogenic con- grana preparations also retain some plastocyanin, tinuity, and so are their photosytsems. According to what is less dependent on the presence of sucrose. this view components functioning in photosystem I Under optimal conditions more plastocyanin is found activities should be spread out over the whole lamel- in the stroma lamellae than in the grana fractions, lar system. It was of interest, therefore, to investi- as was first observed by BASZYNSKI et al. 4. gate the distribution of plastocyanin among the Our values are in contrast to various data in the grana and stroma lamellae. Both, French-press 20 and literature. Especially ARNON and coworkers found digitonin treatment9 can be employed for the sepa- only minimal amounts of plastocyanin left in their ration of these membrane regions3. The data are preparations of stroma lamellae 21, 22. They took this summarized in Table 2. It is clearly seen that a high observation as a support for their model of photo- amount of plastocyanin is retained by the stroma synthesis 23, in which plastocyanin should mediate membrane vesicles after French-press; and even electrons in photosystems driven by short wave more after digitonin treatment, as already previously length light and be concentrated in the grana re- mentioned10. The presence of sucrose at isotonic gions. concentration during fractionation is necessary to In Table 3 evidence is presented that a high sti- keep plastocyanin in the membrane vesicles. The mulation of electron transport by plastocyanin does not always mean a low content of plastocyanin in Treatment of chloroplast plastocyanin content chloroplast membrane preparations, and vice versa. chloroplasts fraction nmoles per mg chlorophyll To observe the stimulation by plastocyanin we measured ascorbate photooxidation without an additio- + 0.4 M sucrose during treatment nal dye as wasfirst describe d by DAVENPORT 24. An — broken chloroplasts 4.5 4.1 inverse correlation between plastocyanin dependent French-press stroma lamellae 1.0 1.7 electron flow and plastocyanin content is indeed ob- grana 1.3 1.5 digitonin stroma lamellae 1.0 3.5 served when chloroplasts are compared with vesicles grana 1.9 1.9 obtained by sonication or French-press fragmenta-

Table 2. The distribution of plastocyanin in the chloroplast tion. A similar trend is found within one method lamellar system. Chloroplasts and membrane fractions were of chloroplast treatment but does no longer hold if prepared and treated for plastocyanin estimation as described membrane vesicles from different treatments are under Methods. The reaction mixture for plastocyanin assay is given in Table 1. compared.

Treatment chloroplast fraction ascorbate plastocyanin photooxidation content

/nmoles 02 uptake nmoles per per mg chlorophyll mg chlorophyll and hr _ broken chloroplasts 20 3.5 French-press stroma lamellae 130 0.6 " +0.4 M sucrose 85 1.7 digitonin 670 1.0 " +0.4 M sucrose 580 2.5 heptane total membrane fraction 35 0.5 ultra sound 170 0.3 " +100 ,MM plastocyanin 170 3.2

Table 3. The lack of correlation between plastocyanin content and plastocyanin stimulation of electron flow. Chloroplast membrane preparations, the assay for plastocyanin and ascorbate photooxidation were carried out as described under Methods. The reaction mixture for the assay of plastocyanin (second column) is given in Table 1. To observe the maximal rates of plastocyanin dependent ascorbate photooxidation by the various chloroplast preparations, 3 nmoles of plastocyanin were added andNADP plus ferredoxin and ferredoxin-NADP reductase were replaced by 0.1 mM methylviologen. The chlorophyll concentration was 10 ug. Oxygen uptake was measured polarographically (first column). Eventual rates without plastocyanin were substracted. 936 P. V. SANE AND G. A. HAUSKA

Class II chloroplasts show almost no stimulation the case of French-press treatment. This observation of electron transport by plastocyanin. Any fragmen- can be explained by the assumption that the reaction tation will liberate plastocyanin and induce the proper of plastocyanin with photosystem I has its plastocyanin stimulation, but to various extents. optimum around pH 8. In French-press preparations Stroma lamellae prepared by passage through the — probobly even more so in chloroplasts — nega- French-press revealed an appreciable electron trans- tive surface charges hinder the also negatively charg- port rate, but those prepared by incubation with ed plastocyanin from reacting. These surface charges, digitonin were much more active (cf. to Ref. 10) possibly in form of proteins or lipids, are removed though containing a higher amount of intrinsic by digitonin and become neutralized to some extent plastocyanin. The corresponding grana fractions at lower pH. were less active but had the same trend (not shown). In contrast, heptane treated chloroplasts having lost almost all their plastocyanin showed very little ac- Discussion tivity. This lack of correlation is most clearly seen when preparations sonicated in the presence or ab- The liberation of plastocyanin, it seems to us, is sence of plastocyanin are compared. Differing in dependent on making the chloroplast membrane plastocyanin content nearly by a 100% they both fluid or leaky. In the case of Triton X-100 treatment show a good rate of plastocyanin stimulated elec- the compartimentation might by permanently lost. tron flow. From these observations we conclude In the other cases of treatment a vesicular structure that other factors than plastocyanin content are reforms, as known form electromicrographs of pre- dominating the secondary interaction of this protein parations obtained with ultra sound 2 or digitonin 10. with the chloroplast membrane. One of these could The amount of plastocyanin liberated wrould then be be the surface charge of the membrane. This idea dependent on the time of transient leakiness. In that gains support from the different pH-profiles found context it interests that plastocyanin can also be re- for plastocyanin-mediated electron flow in digitonin solved by freezing and thawing chloroplasts, even in and French-press preparations (Fig. 2). At pH 6 the the presence of sucrose (cf. to Ref. 25). EDTA-treatment does not remove plastocyanin. Therefore, if "holes" are formed in the membrane by the detach- £ 600_ ment 26 of coupling factor I this is not facilitating 2. the liberation of plastocyanin. > 500. The location of plastocyanin has to be discussed I ^00. on two levels of membrane organization, i. e., mem- £ brane topography and membrane morphology. The first describes how the membrane is built from in- £ 30°- 200. dividual molecules, the second how the membrane 0) 4oc is differentiated into structures of higher order. 100. In a previous publication5 immunological evi- & dence was presented to show that plastocyanin is located at a place in the chloroplast membrane not 5.5 6.0 65 7.0 7.5 8.0 8.5 9.0 pH ^ accessible from the surrounding medium. We suggested that its site is on the inner surface of the Fig. 2. The pH-optima of ascorbate photooxidation in stroma lamellae prepared by French-press or digitonin treatment. membrane in some equilibrium with the thylakoid Ascorbate photooxidation, mediated by excess plastocyanin, space. This suggestion was based on three facts. was measured with methylviologen as electron acceptor as First of all, plastocyanin is a hydrophilic protein, described under Methods and in Table 3. The pH was main- tained by 50 MM MES-NaOH, at pH 5.5 and 6.0, by 50 MM secondly it is rapidly resolved into aqueous media HEPES-NaOH, from pH 6.5 to 7.5, and by 50 MM Tricine- by certain treatments and thirdly this resolution can NaOH, from pH 8.0 to 9.0. be a reversible process. rates are comparable in both preparations. Rising To this view we now can add some data for the the pH to 8 an increase of the rate is found in the location of plastocyanin on the level of membrane digitonin preparations while a decrease occurs in morphology. An intact chloroplast is surrounded by PLASTOCYANIN IN FRAGMENTED CHLOROPLASTS 937 two membranes. The inner membrane is highly dif- tonin would remove all of the plastocyanin. This ferentiated by invaginations, like bacterial or mito- assumption is based on the finding that stimulation chondrial inner membranes. On a cross section this of ascorbate photooxidation by plastocyanin in organization yields three compartments for the photosystem I increases termendously after incuba- chloroplast, the space between the outer and inner tion with digitonin. In complex reaction systems, membrane, the stroma space enclosed by the inner like a vesicular membrane preparation, the straight membrane and the interior of the individual thyla- forward conclusion, that requirement for a compo- koids throught to be interconnected within the la- nent means deficiency, might be wrong. We previ- mellar system. The thylakoid spaces might also be ously presented evidence that plastocyanin added to in connection with the intermembrane space (cf. to vesicular membrane preparation reacts at an arti- Ref. *). From all the known facts we can exclude ficial site different from its original location 5. The that plastocyanin is located in the stroma. It is also unpleasant consequence of this finding is that all not found in the intermembrane space, because this the literature concerning reconstitution of chloro- would be opened up by osmotic shock of class I and plast activities with plastocyanin should be re- also during the preparation of class II chloroplasts. evaluated. Fortunately the reactions of plastocyanin In both instances no plastocyanin is lost to the me- kept at its original place seem to resemble the older dium. So, the intermembrane space is either too data with externally added plastocyanin as far as small to contain enough plastocyanin for detection, electron transport is concerned. Showing a lack; of what is probably not the case 27, or it does not con- correlation between plastocyanin content and elec- tain plastocyanin. If it does not contain plasto- tron transport stimulated by readdition of plastocyanin it, nevertheless, still could be in continuity cyanin (Table 3) we have substantiated once more with the thylakoid space. Then, of course, we have that the secondary effect of this protein needs not to assume that plastocyanin is fixed in some way to reflect its function in vivo. the thylakoid space. Within the lamellar system we The extent of electron flow stimulated by added find plastocyanin in the grana as well as in the plastocyanin increases when isolated chloroplasts stroma lamellar fraction and the latter tends to be are mechanically broken, but is still higher in deter- enriched in plastocyanin on a chlorophyll basis. This gent treated chloroplasts. Fragmentation of the la- corresponds to the distribution of photosystem I mellar system, especially by detergents, seems to activities 3' 9 and suggests that plastocyanin is bound make photosystem I accessible for plastocyanin from to photosystem I (an alternative explanation might outside. In this sense the extent of electron flow be that the grana membranes are less stable i. e. mediated by external plastocyanin could be taken leakier during isolation). as an inverse index for membrane integrity. These and our previous results 5 do not support On of us (P. V. SANE) has been supported by a fel- ARNONS proposed function and location of plasto- lowship from the EMBO during his research in Ger- cyanin 22. many. We thank Dr. J. D. SCHWENN for preparing Similar to us BASZINSKI et al. 4 observed a large class I chloroplasts and Miss. U. FEHRING for measuring the pH-optima in Fig. 2. We gratefully acknowledge portion of plastocyanin remaining in stroma lamel- stimulating discussions with Dr. G. WILDNER and Dr. lae prepared by French-press treatment. They as- A. TREBST, and the experimental assistance of Miss A. sume, however, that subsequent treatment with digi- HELLMICH.

1 R. B. PARK and P. V. SANE, Annu. Rev. Plant Physiol. 22, 7 W. COCKBURN, D. A. WALKER, and C. W. BALDRY, Bio- 395 [1971]. chem. J. 107, 89 [1968]. 2 G. JACOBI and H. LEHMANN, Z. Pflanzenphysiol. 59, 437 8 G. A. HAUSKA and P. V. SANE, Z. Naturforsch., in press. [1968]. 9 J. M. ANDERSON and N. K. BOARDMAN, Biochim. biophy- 3 P. V. SANE, D. J. GOODCHILD, and R. B. PARK, Biochim. sica Acta [Amsterdam] 112, 403 [1966]. biophysica Acta [Amsterdam] 216, 162 [1970]. 10 G. A. HAUSKA, R. E. MCCARTY, and E. RACKER, Biochim. 4 T. BASZYNSKI, J. BRAND, D. W. KROGMANN, and F. L. biophysica Acta [Amsterdam] 197, 206 [1970]. CRANE, Biochim. biophysica Acta [Amsterdam] 234, 537 11 E. ELSTNER, E. PISTORIUS, P. BÖGER, and A. TREBST, [1971]. Planta 79,146 [1968]. 5 G. A. HAUSKA, R. E. MCCARTY, R. J. BERZBORN, and E. 12 M. M. ANDERSON and R. E. MCCARTY, Biochim. biophy- RACKER, J. biol. Chemistry 246, 3524 [1971]. sica Acta [Amsterdam] 189,193 [1969]. 6 R. E. MCCARTY and E. RACKER, J. biol. Chemistry 242, 13 D. I. ARNON, Plant Physiol. 24, 1 [1949]. 3435 [1967]. 938 G. A. HAUSKA AND P. V. SANE

14 S. KATOH, I. SUGA, I. SHIRATORI, and A. TAKAMYIA, Arch. sis", p. 113, (K. SHIBATA, A. TAKAMYIA, A. T. JAGEN- Biochem. Biophysics 94, 136 [1961], DORF, and R. C. FULLER, editors), University of Tokyo 15 S. KATOH and A. SAN PIETRO, in: "The Biochemistry of Press, Tokyo 1967. Copper", p. 407, (J. PEISACH, P. AISEN, and W. E. BLUM- 22 D. I. ARNON, R. K. CHAIN, B. D. MCSWAIN, H. T. TSUJI- BERG, editors), Academic Press, New York 1966. MOTO, and D. B. KNAFF, Proc. nat. Acad. Sei. USA 67. 16 J. S. C. WESSELS, Biochim. biophysica Acta [Amsterdam] 1404 [1970]. 126, 581 [1966]. 23 D. B. KNAFF and D. I. ARNON, Proc. nat. Acad. Sei. USA 17 L. P. VERNON, E. R. SHAW, and B. KE, J. biol. Chemistry 64, 715 [1969]. 241, 4101 [1966]. 24 H. E. DAVENPORT, in: "Non-Heme-Iron Proteins, Role in 18 M. PLESNICAR and D. S. BENDALL, Biochim. biophysica Energy Conversion", p. 115, (A. SAN PIETRO, editor), Acta [Amsterdam] 216, 192 [1970]. Antioch. Press, Yellow Springs, Ohio 1965. 19 A. T. JAGENDORF and E. URIBE, Brookhaven Symp. Biol. 25 U. HEBER, L. TYANKOVA, and K. A. SANTARIUS, Biochim. 19,215 [1966]. biophysica Acta [Amsterdam] 241, 578 [1971]. 20 J. MICHEL and M. MICHEL, Carnegie Inst. Year Book 67, 26 R. E. MCCARTY and E. RACKER, Brookhaven Symp. Biol. 508 [1968]. 19,202 [1966]. 21 D. I. ARNON, J. Y. TSUJIMOTO, B. D. MCSWAIN, and R. K. 27 H. W. HELDT and F. SAUER, Biochim. biophysica Acta CHAIN, in: "Comparative Biochem. Biophys. Photosynthe- [Amsterdam] 234,83 [1971].

The Distribution of Photosynthetic Reactions in the Chloroplast Lamellar System II. Latent ATPase, Proton Pump, Cyclic Phosphorylation and its Sensitivity towards Ammonia

G. A. HAUSKA and P. V. SANE * Abteilung für Biologie, Ruhr-Universität Bochum

(Z. Naturforsch. 27 b. 938—942 [1972] ; leeeived May 15, 1972)

Chloroplast fragmentation, ATPase, Proton pump, Cyclic Phosphorylation, NH4Cl-uncoupling

Cyclic phosphorylation and latent ATPase follow the distribution of photosystem I in the chloroplast membrane. Both activities are found higher in stroma lamellae than in grana. The extent of proton uptake is found to be higher in the grana. Since this uptake depends on internal volume and buffer capacity besides proton pump activity, the distribution of the proton pump proper remains to be elucidated. Any fragmentation of the large inner compartment of the chlorolast lamellar system into smaller vesicles results in decreased sensitivity of cyclic phosphorylation to uncoupling by ammonium chloride. Consequently both, isolated grana and stroma lamellae, show decreased uncoupling by ammonium chloride. The effect can be explained by the action of a membrane potential in photophosphorylation which builds up during illumination and might be more stable in a system with the larger number of individual compartments just for statistical reasons. No assumption on changes in specific ion permeabilities during fragmentation of chloroplasts are needed.

The distribution of energy dependent reactions In this paper we investigate the distribution of and of components, which catalyze them, in an en- coupling factor 1 **, measured as latent ATPase3, ergy transducing membrane system bears on the of cyclic photophosphorylation and of the proton mechanism of energy conservation. The chemical pump between preparations of grana and stroma mechanism 1 requires a close neighbourhood of elec- lamellae, in comparison to the whole chloroplast tron- and energy transfer, while an electrochemical lamellar system. We will show that there is some mechanism2 does not. The electrochemical mecha- proton uptake in every membrane fraction as long nism, on the other hand, postulates an obligate pro- as there is an appreciable rate of phosphorylation. ton pump for phosphorylation. Proton pump and R. E. MCCARTY first observed that ammonium phosphorylation should not be separable by fractio- chloride is a poor uncoupler of photophosphoryla- nation of the membrane system. tion in subchloroplast vesicles, in contrast to chloro-

Requests for reprints should be sent to Dr. G. HAUSKA, ** Abbreviations used: Tricine for (Ar-Tris-hydroxymethyl)- Lehrstuhl für Biochemie der Pflanzen. Abteilung Biologie, methyl)-glycin, MES for 2-(A,morpholino) -ethane sulfonic D-4630 Bochum-Querenburg, Postfach 2148. acid, PMS for (A-methyl)-phenazonium methosulfate, CF, * Present adress: Shraddhanand Peth, Post Ajami, Nagpur/ for coupling factor 1. India.