ARCHIVES
OF BIOCHEMISTRY
AND
A Fluorimetric
168, 266-272 (1975)
BIOPHYSICS
Study of Mg 2f-lnduced Thylakoid
JOAN Nuclear
ISAAKIDOU
Membrane AND
Research Center “Democritus,”
GEORGE Department
Received November
Structural
Changes in
Protein PAPAGEORGIOU of Biology,
Athens,
Greece
12, I974
Low concentrations of Mg’+ (concn < 10 mM) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium l-anilinonaphthalene-Gsulfonate. The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium l-anilinonaphthalene-8-sulfonate to the extent of 126 + 13 nmol/mg protein, and with a dissociation constant KD = 167 pM. Bound ANS fluoresces at 458 nm with a quantum yield Cp = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency Z’,,,., = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation R = 17.3 A is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity. Mg*+ binds to the delipidated membranes with a dissociation constant K, = 2 mM. The binding is attended by enhancement of magnesium I-anilinonaphthalene&sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg*+ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium l-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.
It has been amply documented in recent years that metal cations, especially the divalents, exert specific effects on the properties of inner chloroplast membranes (for a review, see Ref. 1). Such effects are expressed both at the level of electronic excitation, where cations are known to stimulate the Chl a fluorescence of photosystem II and to suppress that of photosystern I (2-6), and at the level of the redox reactions, where they stimulate photosynthetic electron transport (7-9) and coupling to the energy-conserving mechanism (10). One way by which metal cations are supposed to act is by generating structural changes in the thylakoid membrane, which houses the pigments and the enzymatic activities of the photosynthetic apparatus. These changes become evident in several macroscopically measured properties, such
as the turbidity of membrane suspensions and the amount of bound ANS’ fluorescence (ll), the permeability of membranes to protons (12), and the permeability to lipid-soluble nitroaromatic compounds (13, 14). Although the binding characteristics for several metal cations have been reported (15), it is not known whether they bind on negative sites of the protein or lipid moieties of the membrane. In the present paper, we make an attempt to assess the effect of Mg2+ on the structure of the thylakoid protein alone. We use lipid-depleted inner chloroplast membranes, labeled with the fluorochrome ANS. With this system, we study the effect of Mg*+ on such structure’ Abbreviations: ANS, Magnesium l-anilinonaphthalene-8sulfonate; BSA, bovine serum albumin; NaDodSO,, sodium dedecyl sulfate; Tris, tristhydroxymethyllaminomethane. 266
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
STRUCTURAL
CHANGES
related parameters as the fluorescence yield of tryptophyl residues and of bound ANS, as well as the efficiency of excitation energy transfer from tryptophyls to ANS. MATERIALS
AND
METHODS
Envelope-free chloroplasts from market spinach were isolated in high salt according to Murakami and Packer (111, and they were depleted of their lipids as described by Fleischer et al. (16). This method involves exhaustive extraction of the chloroplasts at 4°C with 80% aqueous acetone, containing a trace of ammonia, and produces a reddish residue consisting of membrane protein, plus some carotenoids and plastoquinones (17). The extracted membranes were washed twice with Tris .HCl, 2 mM, sucrose, 250 mM, pH 7.4, and they were stored at -20°C until used. NaDodSO,-acrylamide gel electrophoresis was carried out essentially as described by Weber and Osh m (18). The residue of the acetone extraction was solubilized by suspending it overnight at room temperature in a solution of 10 mM sodium phosphate, pH 7.0, 1% NaDodSO,, and 1% P-mercaptoethanol (19). The ratio of NaDodSO, to protein was 5. Of the solubilized material, 100 pg were applied to the polyacrylamide gel, whose dimensions were 0.65 x 7.0 cm, and electrophoresis was performed at 10 mA per gel for 5 h, with the positive electrode in the lower chamber. All the applied material entered the gel. The gels were stained and fixed in 0.25% Coomassie brilliant blue in methanol-acetic acid-water (5:1:5; volume ratios) overnight, and they were destained with a mixture of 30% methanol and 7% acetic acid. Densitometric patterns of the gels were obtained by scanning at 600 nm in a Joyce-Loebl Chromoscan. Fluorimetric measurements were performed with membranes suspended in the low-salt Tris-sucrose buffer. The samples were incubated with the added MgCl, and ANS for 10 min at room temperature. This was adequate for stabilized fluorescence signals. Tryptophyl fluorescence, and tryptophyl-sensitized ANS fluorescence, were measured with an AmincoBowman spectrofluorometer using excitation and observation bandwidths of 3-4 nm. Directly excited (360-390 nm) ANS fluorescence was measured with a laboratory-built instrument which has been described elsewhere (14), using observation bandwidths of 4-6 nm. Appropriate filters protected the entrance slit of the measuring monochromator against stray excitation light, and the sample against the heat radiation of the excitation source. Further spectroscopic details are given in the legend to the figures. The fluorescence spectra are shown uncorrected for the spectral response of the fluorometers. In estimating the maximal transfer efficiency from tryptophyls to ANS (cf. Fig. 3), the tryptophyl values have been corrected for the inner filter attenuation of the excita-
OF THYLAKOID
PROTEIN
267
tion and fluorescence by the dye, taking into account the 90” angle between excitation and fluorescence detection in the Aminco-Bowman Spectrofluorometer. The employed method is an adaptation of that developed by Duysens (20). The fluorescence quantum yield of ANS bound on acetone-extracted thylakoids was measured by the relative method of Parker and Rees (211, using recrystalized quinine sulfate (Q = 0.550) as the fluorescence standard. The dissociation constant and the number of ANS-binding sites (expressed in nmoles/mg protein) were obtained indirectly, from the fraction of the unbound dye in the supernatant, after precipitating the membranes by centrifugation at 27,OOOgfor 1 h. The supernatant concentration of ANS was determined both by measuring the absorption at 360 nm (e = 4.95 mM-’ cm-‘; Ref. 22), and by means of fluorimetric titration with saturating amounts of BSA
(23). Protein was measured according to Lowry et al. (24). All measurements were made at room temperature. RESULTS
Acetone-extracted chloroplasts retain the thylakoid and grana structures, but the thylakoid membranes appear swollen (25). Figure 1 shows the polypeptide profile of this material as resolved by NaDodSO,acrylamide gel electrophoresis. The profile is closely similar to that reported by Klein and Vernon (19) for the structural protein of spinach chloroplast lamellae. On the basis of this similarity, we may assign molecular weights of 23 and 60 kdaltons to the major polypeptide bands of Fig. 1. The
MIGRATION
Fro 1. Densitometric pattern of polyacrylamide gel, after electrophoresis of acetone-extracted spinach chloroplasts that have been solubilized with 1% NaDodSO,. Protein per gel, ca. 100 pg. Migration is from the right (anode) to the left (cathode). More details are given in Materials and Methods.
268
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AND PAPAGEORGIOU
extrinsic fluorochrome ANS, which binds very poorly to intact lamellae, binds on the acetone-extracted material with a dissociation constant of KD = 167 pM, and a binding number of n = 126 i 13 SD nmol/mg protein. The binding is associated with the appearance of ANS fluorescence with maximum at 458 nm, and a quantum yield Q, = 0.121. Figure 2 presents uncorrected fluorescence spectra, obtained with acetoneextracted lamellae, in the absence (A) and in the presence of increasing amounts of ANS (B-E). When the dye is absent, fluorescence is emitted by the tryptophyl residues only, with a band maximum at 345 nm. The position of this maximum is not shifted when the excitation band is moved from 280 to 300 nm. This property, as well as the complete absence of tyrosine fluorescence (emission maximum at 310 nm; Ref. 26) from the spectra of Fig. 2 indicate a quantitative transfer of tyrosyl excitation to tryptophyls. In the presence of ANS, tryptophyl fluorescence becomes quenched, while the fluorescence of membrane-bound ANS (at 458 nm) makes its appearance. Although
01 0
008
004 ANS
, @4-’
FIG. 3. Double-reciprocal plot of the decrease of tryptophyl fluorescence against the concentration of ANS. F,, and F denote tryptophyl fluorescence in the absence, and in the presence of the dye. Excitation, 300 nm; observation, 344 nm; half-band widths, 3-4 nm. The plotted points have been corrected for the inner filter attenuation of the exciting light and of the fluorescence signal by the added ANS. Protein content, 85 Mg/ml
part of the exciting light is absorbed by the free dye, the latter does not fluoresce. Consequently, the 458-nm fluorescence band represents tryptophyl-sensitized emission from membrane-bound ANS. The existence of an isoemissive point (here at 410 nm; cf. Fig. 2), whose spectral position is independent of the concentration, has been shown to evidence excitation energy transfer (27). The efficiency of excitation transfer (T) can be estimated from the decrease of the tryptophyl fluorescence yield in the presence of ANS, since T = (a,, - a)/+,,, where ‘P,,and + stand for the donor fluorescence yields in the absence and in the presence of the added acceptor (26). As shown in Fig. 2, the position of the tryptophyl fluorescence maximum (344 nm) is not shifted in the presence of the dye. This permits the substitution of the fluorescence intensities at the maximum for the quantum yield values in the expression for T. Figure 3 shows that plots of T-' = Fd(Fo 600 500 290 300 F) against the reciprocal ANS concentraWAVELENGTH nm tion are linear, allowing extrapolation to FIG. 2. Fluorescence spectra of acetone-extracted the ordinate axis. The inverse of the ordithylakoid membranes. A, with no additions; B, C, D, nate intercept gives the maximal transfer and E with 5 PM ANS, 10 pM ANS, 15 PM ANS, and 20 efficiency (T,,,,,), which corresponds to the PM ANS, respectively. Excitation, 280 nm. Half-band widths of excitation and observation, 3-4 nm. The state of complete occupation of all the spectra are shown uncorrected for the spectral varia- ANS-binding sites of the membrane (28). In this case, Fig. 3 corresponds to Z’,,,,, = tion of the detection sensitivity of the Aminco-Bowman Fluorimeter. 0.85. For these calculations, we used emis-
STRUCTURAL
CHANGES
sion spectra like those of Fig. 2, but excited at 300 nm where the difference between the absorptivities of tryptophan and ANS is maximal. This minimizes the correction factors needed to correct for inner filter absorption of the excitation and of the tryptophyl fluorescence by the added dye. When MgCl, is added to suspensions of acetone-extracted chloroplast membranes, the tryptophyl fluorescence is suppressed, while the directly excited fluorescence of bound ANS is stimulated. Double-reciprocal plots of these changes are shown in Fig. 4. The increase of ANS fluorescence and the decrease of tryptophyl fluorescence as a function of the concentration of added Mg2+ were measured independently. Bound ANS was excited directly at 360-390 nm, where tryptophan does not absorb, while the decrease of tryptophyl fluorescence with the added Mg2+ was measured in the absence of ANS. In this way, possible effects of Mg2+ on the excitation transfer rate are excluded from the recorded changes. In addition, the direct excitation ensures that we take into account the entire population of the bound
! /
F-F.
OF THYLAKOID
269
PROTEIN
PROTEIN,
mlipg
FIG. 5. Double-reciprocal
plots of bound ANS fluorescence against the protein concentration of suspensions of acetone-extracted thylakoid membranes. ANS, 20 PM; MgCl,, 10 MM; MnCl,, 10 pM. ANS fluorescence was excited and measured as described in Fig. 4.
dye, while sensitized excitation would be selective for the fraction of dye molecules that are proximal to the tryptophyl residues. Figure 4 indicates that Mg2+ modifies the fluorescence yields of both chromophores (Try and ANS) by binding to the membrane with a dissociation constant K. = 2 mM. This may suggest that it is the same binding site that influences both chromophores. Other divalent metal cations (Ca’+, Mn’+) were found to bind with the same dissociation constant and to cause the same effects on the fluorescence spectra of ANS-labeled thylakoid protein as Mgz+. On the other hand, K+ and Na+ cause smaller changes that correspond to
KD = 100 mM. MgCI,,
mM-’
FiG. 4. Double-reciprocal plots of the decrease of tryptophyl fluorescence and the increase of bound ANS fluorescence against the concentration of MgCl, in a suspension of acetone-extracted thylakoid membranes. F,, and F, the fluorescence of each chromophore in the absence and in the presence of MgCl,, respectively. The fluorescence of ANS was excited at 360-390 nm (Corning glass filter C.S. 7-51, and 5 cm of 1% CuSO,) and was observed at 480 nm, with a half-band width of 4-6 nm. Tryptophyl fluorescence was measured in the absence of ANS at 344 nm, with excitation at 300 nm.
The cation-induced enhancement of ANS fluorescence can be attributed to an increase of the fluorescence quantum yield of the bound dye, to increased binding, or even to a combination of these factors. Figure 5, which displays double-reciprocal plots of bound ANS fluorescence against the concentration of membrane protein, shows that, indeed, metal cations lead to a higher ANS fluorescence yield. Since at infinite protein all the available dye is bound, the reciprocals of the ordinate intercepts are proportional to the quantum yield of bound ANS fluorescence in each
270
ISAAKIDOU
AND PAPAGEORGIOU
particular case. On the basis of the yield determined in the absence of metal cations @ = 0.121, we may calculate yields of 0.133 and 0.196 in the presence of saturating amounts of Mgz+ and Mn2+. Since ANS is known to be more fluorescent in hydrophobic media (29), these results suggest a cation-induced increase of the membrane hydrophobicity in the vicinity of the ANS binding site. The effect of increasing concentrations of Mg2+ on the efficiency of excitation energy transfer from the tryptophyls to the ANS is illustrated in Fig. 6, which displays the cation-induced decrease of tryptophyl fluorescence in the absence and in the presence of 10 PM ANS. In the absence of Mgz+ the fluorescence intensities have been normalized to 100, so that the difference between the two concentration curves can be related directly to the cation effect on the transfer efficiency. According to this figure, most of the Mg2+ effect on tryptophyl fluorescence is unrelated to the change in the transfer efficiency, consisting in its greatest part of a decrease in the fluorescence yield which is independent of the presence of the acceptor chromophore. These data correspond to cation stimulation of the transfer efficiency of about 2% only.
0
2
4 MgC12
6
8
10
,mM
FIG. 6. The decrease of tryptophyl fluorescence as a function of the concentration of MgCll in suspensions of acetone-extracted thylakoid membranes, in the absence and in the presence of 10 PM ANS. The fluorescence signals in the absence of added MgCl, (F,) are normalized to 100. Tryptophyl fluorescence was excited and measured as described in Fig. 3.
DISCUSSION
Structural and structure-related functional changes of thylakoid membranes, induced by Mg2+ and other divalent metal cations, have been investigated by several groups (l-15), but the exact effects of these cations on the lipid and protein moieties of the membrane remain largely unclear. Furthermore, it is not known to what extent the conformation of individual membrane proteins is affected during cation-induced gross morphological changes of chloroplasts, such as the stacking of thylakoids into grana (11, 30). We have attempted, here, to gather information relevant to these questions by studying the effect of Mg2+ on the fluorescence properties of a native membrane chromophore, the tryptophyl residue, and of an externally added label, ANS. Thylakoid membranes extracted with 80% acetone show little disaggregation (25), and consist essentially of structural protein monomers (23 K) and aggregates (60 K; cf. Fig. 1). The external fluorescent label ANS binds to these membranes with a dissociation constant KD = 167 FM, and a binding number n = 126 * 13 SD nmoles/mg protein. Two properties characterize the ANS-binding site: First, its low polarity, as deduced from the high absolute quantum yield, @ = 0.121, of bound ANS fluorescence. On the basis of the quantum yield values for ANS fluorescence in alcohol-water mixtures, given by Stryer (31), the polarity of this site approximates that of a 78% aqueous solution of alcohol. Second, its proximity to the tryptophyl residues of the membrane protein, as deduced from the high efficiency of excitation energy transfer from tryptophyl to ANS (T,,, = 0.85), when all the ANS-binding sites are occupied. It can be calculated from the data reported by P. Weber (32) that 1 mg of thylakoid structural protein from spinach contains 184 nmoles of tryptophan. Accordingly, the ANS-binding number obtained here may imply that only two-thirds of the membrane tryptophyls are proximal to the ANS binding sites. In view of our
STRUCTURAL
CHANGES OF THYLAKOID
complete ignorance about the molecular orientations of these chromophores, it is impossible to estimate unambiguously the interchromophore separation from the calculated efficiency of excitation energy transfer. Brocklehurst et al. (23), assuming completely random orientation of tryptophyls and ANS in mitochondrial particles, calculated an interchromophore distance R, = 23.3 A for 50% energy transfer probability. Taking this R, to be typical of chloroplast membranes also, we may use the relation T,,,,, = R,V(R,” + R’) (29 to derive an indicative value R = 17.3 A for the separation between the ANS-binding site and the vicinal tryptophyl residue. As it can be inferred from the increase of ANS fluorescence, and the decrease of tryptophyl fluorescence, acetone-extracted thylakoid membranes experience structural changes in the neighborhood of the ANS-binding site, when Mgz+ is added to their suspensions. Both fluorescence changes saturate at about 5 mM MgCl,. The dissociation constant of the complex Mg’+-membrane site, calculated from the respective concentration curves, is in both cases K. = 2 mM (Fig. 4). This coincidence points to the existence of a single Mgz+binding site, whose occupation influences the fluorescence characteristics of both chromophores. Other divalent cations, such as Ca2+ and MnZ+, were found to bind at the same site (as deduced from the similar effects they exert on the fluorescence of tryptophyl and of ANS), and with the same dissociation constant as Mgz+. Using intact envelope-free chloroplasts, suspended in a buffer essentially similar to the one employed here (i.e., very low in salt), Gross and Hess (15) identified two different binding sites for the divalent cations Mg2+, Ca2+, and Mn2+, with dissociation constants KD = 8 f 3 PM and K. = 51 * 8 pM. Our evidence is not sufficient for an unequivocal correlation of the divalent cation binding site, found here with delipidated lamellae, with one of those of intact lamellae. Indeed, in view of the widely different dissociation constants, one may suggest that we see here an entirely
PROTEIN
271
new site that has been unmasked by the removal of the membrane lipids. If this is true, then both sites reported by Gross and Hess (15) should be assigned to the lipid moiety of the lamella. Alternatively, one may speculate that we observe here the weaker binding site (KD = 51 =L 8 PM), but that a structural change, caused by the acetone treatment, has shifted the equilibrium toward less binding (to K. = 2 mM). In such a case, only the stronger binding site of intact lamellae (KD = 8 h 3 mM) should be assigned to a lipid-associated nucleophile. The fact that the binding site is common for all divalents tested, both in intact membranes (15), as well as in delipidated membranes, lends some support to the second hypothesis. As a result of structural changes induced by metal cations, the neighborhood of the ANS-binding site becomes more hydrophobic. This is evidenced by the increased fluorescence yield of membrane-bound ANS in the presence of Mg2+ and Mn2+ (Fig. 5). The decrease of tryptophyl fluorescence, under the same conditions, should also be correlated to the higher hydrophobicity at the neighborhood of the ANS-binding site, i$ view of the close proximity (R - 17.3 A) of the two chromophores. The fluorescence of tryptophyl residue is known to be unspecific as to its dependence on the polarity of its immediate environment (33). Accordingly, the direction of polarity changes in the neighborhood of membrane tryptophyls can be elucidated only in conjuction with concomitant effects on the fluorescence of a proximal polarity probe, such as the ANS chromophore. Despite the dramatic effects of the divalent metal cations on the fluorescence yields of tryptophyl and of bound ANS, the excitation energy exchange between these two chromophores is only slightly altered (Fig. 6). On the basis of the Rm6 law of resonance excitation transfer (34), one might expect very substantial changes in the transfer rate for even slight changes in the interchromophore separation. One possibility, although remote, is to consider
272
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AND PAPAGEORGIOU
cation-induced changes in both separation and mutual orientation of the interacting chromophores, of such fortuitous magnitudes, as to be mutually compensating. Disregarding the possibility of an orientation change, we may deduce from Fig. 6 gnd on the basis of an assumed R. = 23.3 A, that Mg*+ shortens the distance from ;he tryptophyl to bound ANS by about 0.5 A only. We may, therefore, conclude tentatively that metal divalent cations do not generate short-range structural changes in the neighborhood of the ANS-binding site. REFERENCES 1. PAPAGEORGIOU,G. (ln74) in Bioenergetics of Photosynthesis (Govindjee, ed.) Academic Press, New York. 2. BRODY, S. S., ZIEGELMAIR, C. A., SAMUELS, A., AND BRODY, M. (1966) Plant Physiol. 41,1709-1714. 3. HOMANN, P. (1969) Plant Physiol. 44, 932-936. 4. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. 5. SUN, A. S. K., AND SAUER, K. (1972) Biochim. Biophys. Acta 256, 409-427. 6. MOHANTY, P. K., BRAUN, B. Z., AND GOVINDJEE (1973) Biochim. Biophys. Acta 292, 459-476. 7. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. 8. RURAINSKI, H. J., RANDLES, J., AND HOCH, G. E. (1971) Fed. Eur. Biochem. Sot. Lett. 13, 98-100. 9. MARSHO, T. V., AND KOK, B. (1974) Biuchim. Biophys. Acta 333, 353-365. 10. GROSS, E. L., DILLEY, R. A., AND SAN PIETRO, A. (1969) Arch. Biochem. Biophys. 134, 450-462. 11. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biothem. Biophys. 146, 337-347. 12. DILLEY, R. A., AND SHAVIT, N. (1968) B&him. Biophys. Acta 162, 86-96. 13. PAPACEORGIOU,G. (1971) in Proc. 2nd Int. Congr. Photosynth. Res. (Forti, G., Avron, M., and Melandri, A. eds), Vol. 2, pp. 1535-1544, Dr. W. Jung, Publ., The Hague.
14. PAPACEORGIOU, G., AND ARGOUDELIS, C. (1973) Arch. Biochem. Biophys. 156, 134-142. 15. GROSS, E. L., AND HESS, S. C. (1974) Biochim. Biophys. Acta 339, 334-346. 16. FLEISCHER, S., FLEISCHER, B., AND STOECKENIUS, W. (1967) J. Cell Biol. 32, 193-208. 17. JI, T. H., HESS, T. L., AND BENSON, A. A. (1968) Biochim. Biophys. Acta 150, 676-685. 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 19. KLEIN, S. M., AND VERNON, L. P. (1974) Photothem. Photobiol. 19, 43-49. 20. DWSENS, L. N. M. (1952) PH.D. Thesis, University of Utrecht. 21. PARKER, C. A., AND REES, W. T. (1960) Analyst 85, 587-600. 22. WEBER, G., AND YOUNG, L. B. (1964) J. Biol. Chem. 239, 1415-1423. 23. BROCKLEHURST,J. R., FREEDMAN, R. B., HANCOCK, D. J., AND RADDA, G. K. (1970) Biochem. J. 116, 721-731. 24. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 25. HUANG, J.-S., HUANG, P.-Y., AND GOODMAN, R. N. (1973) Amer. J. Bot. 60, 80-85. 26. BRAND, L., AND WITHOLT, B. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 776-856, Academic Press, New York. 27. ANDERSON, R., AND WEBER, G. (1965) Biochemistry 4, 1949-1957. 28. WALLACH, D. F. H., FERBER, E., SELIN, D., WEIDEKAMM, E., AND FISCHER, H. (1970) Biochim. Biophys. Acta 203, 67-76. 29. TURNER, D. C., AND BRAND, L. (1968) Biochemistry 7, 3381-3390. 30. IZAWA, S., AND GOOD, N. E. (1966) Plant Physiol. 41,533-543. 31. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 32. WEBER, P. (1962) 2. Naturforsch. 17B, 683-688. 33. KRONMAN, M. J., AND HOLMES, L. G. (1971) Photochem. Photobiol. 14, 113-134. 34. F~RSTER, T. (1951) in Fluoreszenz Organischer Verbindungen, pp. 83-86, Vandenhoeck and Ruprecht, GSttingen.
OF BIOCHEMISTRY
AND
A Fluorimetric
168, 266-272 (1975)
BIOPHYSICS
Study of Mg 2f-lnduced Thylakoid
JOAN Nuclear
ISAAKIDOU
Membrane AND
Research Center “Democritus,”
GEORGE Department
Received November
Structural
Changes in
Protein PAPAGEORGIOU of Biology,
Athens,
Greece
12, I974
Low concentrations of Mg’+ (concn < 10 mM) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium l-anilinonaphthalene-Gsulfonate. The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium l-anilinonaphthalene-8-sulfonate to the extent of 126 + 13 nmol/mg protein, and with a dissociation constant KD = 167 pM. Bound ANS fluoresces at 458 nm with a quantum yield Cp = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency Z’,,,., = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation R = 17.3 A is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity. Mg*+ binds to the delipidated membranes with a dissociation constant K, = 2 mM. The binding is attended by enhancement of magnesium I-anilinonaphthalene&sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg*+ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium l-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.
It has been amply documented in recent years that metal cations, especially the divalents, exert specific effects on the properties of inner chloroplast membranes (for a review, see Ref. 1). Such effects are expressed both at the level of electronic excitation, where cations are known to stimulate the Chl a fluorescence of photosystem II and to suppress that of photosystern I (2-6), and at the level of the redox reactions, where they stimulate photosynthetic electron transport (7-9) and coupling to the energy-conserving mechanism (10). One way by which metal cations are supposed to act is by generating structural changes in the thylakoid membrane, which houses the pigments and the enzymatic activities of the photosynthetic apparatus. These changes become evident in several macroscopically measured properties, such
as the turbidity of membrane suspensions and the amount of bound ANS’ fluorescence (ll), the permeability of membranes to protons (12), and the permeability to lipid-soluble nitroaromatic compounds (13, 14). Although the binding characteristics for several metal cations have been reported (15), it is not known whether they bind on negative sites of the protein or lipid moieties of the membrane. In the present paper, we make an attempt to assess the effect of Mg2+ on the structure of the thylakoid protein alone. We use lipid-depleted inner chloroplast membranes, labeled with the fluorochrome ANS. With this system, we study the effect of Mg*+ on such structure’ Abbreviations: ANS, Magnesium l-anilinonaphthalene-8sulfonate; BSA, bovine serum albumin; NaDodSO,, sodium dedecyl sulfate; Tris, tristhydroxymethyllaminomethane. 266
Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
STRUCTURAL
CHANGES
related parameters as the fluorescence yield of tryptophyl residues and of bound ANS, as well as the efficiency of excitation energy transfer from tryptophyls to ANS. MATERIALS
AND
METHODS
Envelope-free chloroplasts from market spinach were isolated in high salt according to Murakami and Packer (111, and they were depleted of their lipids as described by Fleischer et al. (16). This method involves exhaustive extraction of the chloroplasts at 4°C with 80% aqueous acetone, containing a trace of ammonia, and produces a reddish residue consisting of membrane protein, plus some carotenoids and plastoquinones (17). The extracted membranes were washed twice with Tris .HCl, 2 mM, sucrose, 250 mM, pH 7.4, and they were stored at -20°C until used. NaDodSO,-acrylamide gel electrophoresis was carried out essentially as described by Weber and Osh m (18). The residue of the acetone extraction was solubilized by suspending it overnight at room temperature in a solution of 10 mM sodium phosphate, pH 7.0, 1% NaDodSO,, and 1% P-mercaptoethanol (19). The ratio of NaDodSO, to protein was 5. Of the solubilized material, 100 pg were applied to the polyacrylamide gel, whose dimensions were 0.65 x 7.0 cm, and electrophoresis was performed at 10 mA per gel for 5 h, with the positive electrode in the lower chamber. All the applied material entered the gel. The gels were stained and fixed in 0.25% Coomassie brilliant blue in methanol-acetic acid-water (5:1:5; volume ratios) overnight, and they were destained with a mixture of 30% methanol and 7% acetic acid. Densitometric patterns of the gels were obtained by scanning at 600 nm in a Joyce-Loebl Chromoscan. Fluorimetric measurements were performed with membranes suspended in the low-salt Tris-sucrose buffer. The samples were incubated with the added MgCl, and ANS for 10 min at room temperature. This was adequate for stabilized fluorescence signals. Tryptophyl fluorescence, and tryptophyl-sensitized ANS fluorescence, were measured with an AmincoBowman spectrofluorometer using excitation and observation bandwidths of 3-4 nm. Directly excited (360-390 nm) ANS fluorescence was measured with a laboratory-built instrument which has been described elsewhere (14), using observation bandwidths of 4-6 nm. Appropriate filters protected the entrance slit of the measuring monochromator against stray excitation light, and the sample against the heat radiation of the excitation source. Further spectroscopic details are given in the legend to the figures. The fluorescence spectra are shown uncorrected for the spectral response of the fluorometers. In estimating the maximal transfer efficiency from tryptophyls to ANS (cf. Fig. 3), the tryptophyl values have been corrected for the inner filter attenuation of the excita-
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tion and fluorescence by the dye, taking into account the 90” angle between excitation and fluorescence detection in the Aminco-Bowman Spectrofluorometer. The employed method is an adaptation of that developed by Duysens (20). The fluorescence quantum yield of ANS bound on acetone-extracted thylakoids was measured by the relative method of Parker and Rees (211, using recrystalized quinine sulfate (Q = 0.550) as the fluorescence standard. The dissociation constant and the number of ANS-binding sites (expressed in nmoles/mg protein) were obtained indirectly, from the fraction of the unbound dye in the supernatant, after precipitating the membranes by centrifugation at 27,OOOgfor 1 h. The supernatant concentration of ANS was determined both by measuring the absorption at 360 nm (e = 4.95 mM-’ cm-‘; Ref. 22), and by means of fluorimetric titration with saturating amounts of BSA
(23). Protein was measured according to Lowry et al. (24). All measurements were made at room temperature. RESULTS
Acetone-extracted chloroplasts retain the thylakoid and grana structures, but the thylakoid membranes appear swollen (25). Figure 1 shows the polypeptide profile of this material as resolved by NaDodSO,acrylamide gel electrophoresis. The profile is closely similar to that reported by Klein and Vernon (19) for the structural protein of spinach chloroplast lamellae. On the basis of this similarity, we may assign molecular weights of 23 and 60 kdaltons to the major polypeptide bands of Fig. 1. The
MIGRATION
Fro 1. Densitometric pattern of polyacrylamide gel, after electrophoresis of acetone-extracted spinach chloroplasts that have been solubilized with 1% NaDodSO,. Protein per gel, ca. 100 pg. Migration is from the right (anode) to the left (cathode). More details are given in Materials and Methods.
268
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AND PAPAGEORGIOU
extrinsic fluorochrome ANS, which binds very poorly to intact lamellae, binds on the acetone-extracted material with a dissociation constant of KD = 167 pM, and a binding number of n = 126 i 13 SD nmol/mg protein. The binding is associated with the appearance of ANS fluorescence with maximum at 458 nm, and a quantum yield Q, = 0.121. Figure 2 presents uncorrected fluorescence spectra, obtained with acetoneextracted lamellae, in the absence (A) and in the presence of increasing amounts of ANS (B-E). When the dye is absent, fluorescence is emitted by the tryptophyl residues only, with a band maximum at 345 nm. The position of this maximum is not shifted when the excitation band is moved from 280 to 300 nm. This property, as well as the complete absence of tyrosine fluorescence (emission maximum at 310 nm; Ref. 26) from the spectra of Fig. 2 indicate a quantitative transfer of tyrosyl excitation to tryptophyls. In the presence of ANS, tryptophyl fluorescence becomes quenched, while the fluorescence of membrane-bound ANS (at 458 nm) makes its appearance. Although
01 0
008
004 ANS
, @4-’
FIG. 3. Double-reciprocal plot of the decrease of tryptophyl fluorescence against the concentration of ANS. F,, and F denote tryptophyl fluorescence in the absence, and in the presence of the dye. Excitation, 300 nm; observation, 344 nm; half-band widths, 3-4 nm. The plotted points have been corrected for the inner filter attenuation of the exciting light and of the fluorescence signal by the added ANS. Protein content, 85 Mg/ml
part of the exciting light is absorbed by the free dye, the latter does not fluoresce. Consequently, the 458-nm fluorescence band represents tryptophyl-sensitized emission from membrane-bound ANS. The existence of an isoemissive point (here at 410 nm; cf. Fig. 2), whose spectral position is independent of the concentration, has been shown to evidence excitation energy transfer (27). The efficiency of excitation transfer (T) can be estimated from the decrease of the tryptophyl fluorescence yield in the presence of ANS, since T = (a,, - a)/+,,, where ‘P,,and + stand for the donor fluorescence yields in the absence and in the presence of the added acceptor (26). As shown in Fig. 2, the position of the tryptophyl fluorescence maximum (344 nm) is not shifted in the presence of the dye. This permits the substitution of the fluorescence intensities at the maximum for the quantum yield values in the expression for T. Figure 3 shows that plots of T-' = Fd(Fo 600 500 290 300 F) against the reciprocal ANS concentraWAVELENGTH nm tion are linear, allowing extrapolation to FIG. 2. Fluorescence spectra of acetone-extracted the ordinate axis. The inverse of the ordithylakoid membranes. A, with no additions; B, C, D, nate intercept gives the maximal transfer and E with 5 PM ANS, 10 pM ANS, 15 PM ANS, and 20 efficiency (T,,,,,), which corresponds to the PM ANS, respectively. Excitation, 280 nm. Half-band widths of excitation and observation, 3-4 nm. The state of complete occupation of all the spectra are shown uncorrected for the spectral varia- ANS-binding sites of the membrane (28). In this case, Fig. 3 corresponds to Z’,,,,, = tion of the detection sensitivity of the Aminco-Bowman Fluorimeter. 0.85. For these calculations, we used emis-
STRUCTURAL
CHANGES
sion spectra like those of Fig. 2, but excited at 300 nm where the difference between the absorptivities of tryptophan and ANS is maximal. This minimizes the correction factors needed to correct for inner filter absorption of the excitation and of the tryptophyl fluorescence by the added dye. When MgCl, is added to suspensions of acetone-extracted chloroplast membranes, the tryptophyl fluorescence is suppressed, while the directly excited fluorescence of bound ANS is stimulated. Double-reciprocal plots of these changes are shown in Fig. 4. The increase of ANS fluorescence and the decrease of tryptophyl fluorescence as a function of the concentration of added Mg2+ were measured independently. Bound ANS was excited directly at 360-390 nm, where tryptophan does not absorb, while the decrease of tryptophyl fluorescence with the added Mg2+ was measured in the absence of ANS. In this way, possible effects of Mg2+ on the excitation transfer rate are excluded from the recorded changes. In addition, the direct excitation ensures that we take into account the entire population of the bound
! /
F-F.
OF THYLAKOID
269
PROTEIN
PROTEIN,
mlipg
FIG. 5. Double-reciprocal
plots of bound ANS fluorescence against the protein concentration of suspensions of acetone-extracted thylakoid membranes. ANS, 20 PM; MgCl,, 10 MM; MnCl,, 10 pM. ANS fluorescence was excited and measured as described in Fig. 4.
dye, while sensitized excitation would be selective for the fraction of dye molecules that are proximal to the tryptophyl residues. Figure 4 indicates that Mg2+ modifies the fluorescence yields of both chromophores (Try and ANS) by binding to the membrane with a dissociation constant K. = 2 mM. This may suggest that it is the same binding site that influences both chromophores. Other divalent metal cations (Ca’+, Mn’+) were found to bind with the same dissociation constant and to cause the same effects on the fluorescence spectra of ANS-labeled thylakoid protein as Mgz+. On the other hand, K+ and Na+ cause smaller changes that correspond to
KD = 100 mM. MgCI,,
mM-’
FiG. 4. Double-reciprocal plots of the decrease of tryptophyl fluorescence and the increase of bound ANS fluorescence against the concentration of MgCl, in a suspension of acetone-extracted thylakoid membranes. F,, and F, the fluorescence of each chromophore in the absence and in the presence of MgCl,, respectively. The fluorescence of ANS was excited at 360-390 nm (Corning glass filter C.S. 7-51, and 5 cm of 1% CuSO,) and was observed at 480 nm, with a half-band width of 4-6 nm. Tryptophyl fluorescence was measured in the absence of ANS at 344 nm, with excitation at 300 nm.
The cation-induced enhancement of ANS fluorescence can be attributed to an increase of the fluorescence quantum yield of the bound dye, to increased binding, or even to a combination of these factors. Figure 5, which displays double-reciprocal plots of bound ANS fluorescence against the concentration of membrane protein, shows that, indeed, metal cations lead to a higher ANS fluorescence yield. Since at infinite protein all the available dye is bound, the reciprocals of the ordinate intercepts are proportional to the quantum yield of bound ANS fluorescence in each
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ISAAKIDOU
AND PAPAGEORGIOU
particular case. On the basis of the yield determined in the absence of metal cations @ = 0.121, we may calculate yields of 0.133 and 0.196 in the presence of saturating amounts of Mgz+ and Mn2+. Since ANS is known to be more fluorescent in hydrophobic media (29), these results suggest a cation-induced increase of the membrane hydrophobicity in the vicinity of the ANS binding site. The effect of increasing concentrations of Mg2+ on the efficiency of excitation energy transfer from the tryptophyls to the ANS is illustrated in Fig. 6, which displays the cation-induced decrease of tryptophyl fluorescence in the absence and in the presence of 10 PM ANS. In the absence of Mgz+ the fluorescence intensities have been normalized to 100, so that the difference between the two concentration curves can be related directly to the cation effect on the transfer efficiency. According to this figure, most of the Mg2+ effect on tryptophyl fluorescence is unrelated to the change in the transfer efficiency, consisting in its greatest part of a decrease in the fluorescence yield which is independent of the presence of the acceptor chromophore. These data correspond to cation stimulation of the transfer efficiency of about 2% only.
0
2
4 MgC12
6
8
10
,mM
FIG. 6. The decrease of tryptophyl fluorescence as a function of the concentration of MgCll in suspensions of acetone-extracted thylakoid membranes, in the absence and in the presence of 10 PM ANS. The fluorescence signals in the absence of added MgCl, (F,) are normalized to 100. Tryptophyl fluorescence was excited and measured as described in Fig. 3.
DISCUSSION
Structural and structure-related functional changes of thylakoid membranes, induced by Mg2+ and other divalent metal cations, have been investigated by several groups (l-15), but the exact effects of these cations on the lipid and protein moieties of the membrane remain largely unclear. Furthermore, it is not known to what extent the conformation of individual membrane proteins is affected during cation-induced gross morphological changes of chloroplasts, such as the stacking of thylakoids into grana (11, 30). We have attempted, here, to gather information relevant to these questions by studying the effect of Mg2+ on the fluorescence properties of a native membrane chromophore, the tryptophyl residue, and of an externally added label, ANS. Thylakoid membranes extracted with 80% acetone show little disaggregation (25), and consist essentially of structural protein monomers (23 K) and aggregates (60 K; cf. Fig. 1). The external fluorescent label ANS binds to these membranes with a dissociation constant KD = 167 FM, and a binding number n = 126 * 13 SD nmoles/mg protein. Two properties characterize the ANS-binding site: First, its low polarity, as deduced from the high absolute quantum yield, @ = 0.121, of bound ANS fluorescence. On the basis of the quantum yield values for ANS fluorescence in alcohol-water mixtures, given by Stryer (31), the polarity of this site approximates that of a 78% aqueous solution of alcohol. Second, its proximity to the tryptophyl residues of the membrane protein, as deduced from the high efficiency of excitation energy transfer from tryptophyl to ANS (T,,, = 0.85), when all the ANS-binding sites are occupied. It can be calculated from the data reported by P. Weber (32) that 1 mg of thylakoid structural protein from spinach contains 184 nmoles of tryptophan. Accordingly, the ANS-binding number obtained here may imply that only two-thirds of the membrane tryptophyls are proximal to the ANS binding sites. In view of our
STRUCTURAL
CHANGES OF THYLAKOID
complete ignorance about the molecular orientations of these chromophores, it is impossible to estimate unambiguously the interchromophore separation from the calculated efficiency of excitation energy transfer. Brocklehurst et al. (23), assuming completely random orientation of tryptophyls and ANS in mitochondrial particles, calculated an interchromophore distance R, = 23.3 A for 50% energy transfer probability. Taking this R, to be typical of chloroplast membranes also, we may use the relation T,,,,, = R,V(R,” + R’) (29 to derive an indicative value R = 17.3 A for the separation between the ANS-binding site and the vicinal tryptophyl residue. As it can be inferred from the increase of ANS fluorescence, and the decrease of tryptophyl fluorescence, acetone-extracted thylakoid membranes experience structural changes in the neighborhood of the ANS-binding site, when Mgz+ is added to their suspensions. Both fluorescence changes saturate at about 5 mM MgCl,. The dissociation constant of the complex Mg’+-membrane site, calculated from the respective concentration curves, is in both cases K. = 2 mM (Fig. 4). This coincidence points to the existence of a single Mgz+binding site, whose occupation influences the fluorescence characteristics of both chromophores. Other divalent cations, such as Ca2+ and MnZ+, were found to bind at the same site (as deduced from the similar effects they exert on the fluorescence of tryptophyl and of ANS), and with the same dissociation constant as Mgz+. Using intact envelope-free chloroplasts, suspended in a buffer essentially similar to the one employed here (i.e., very low in salt), Gross and Hess (15) identified two different binding sites for the divalent cations Mg2+, Ca2+, and Mn2+, with dissociation constants KD = 8 f 3 PM and K. = 51 * 8 pM. Our evidence is not sufficient for an unequivocal correlation of the divalent cation binding site, found here with delipidated lamellae, with one of those of intact lamellae. Indeed, in view of the widely different dissociation constants, one may suggest that we see here an entirely
PROTEIN
271
new site that has been unmasked by the removal of the membrane lipids. If this is true, then both sites reported by Gross and Hess (15) should be assigned to the lipid moiety of the lamella. Alternatively, one may speculate that we observe here the weaker binding site (KD = 51 =L 8 PM), but that a structural change, caused by the acetone treatment, has shifted the equilibrium toward less binding (to K. = 2 mM). In such a case, only the stronger binding site of intact lamellae (KD = 8 h 3 mM) should be assigned to a lipid-associated nucleophile. The fact that the binding site is common for all divalents tested, both in intact membranes (15), as well as in delipidated membranes, lends some support to the second hypothesis. As a result of structural changes induced by metal cations, the neighborhood of the ANS-binding site becomes more hydrophobic. This is evidenced by the increased fluorescence yield of membrane-bound ANS in the presence of Mg2+ and Mn2+ (Fig. 5). The decrease of tryptophyl fluorescence, under the same conditions, should also be correlated to the higher hydrophobicity at the neighborhood of the ANS-binding site, i$ view of the close proximity (R - 17.3 A) of the two chromophores. The fluorescence of tryptophyl residue is known to be unspecific as to its dependence on the polarity of its immediate environment (33). Accordingly, the direction of polarity changes in the neighborhood of membrane tryptophyls can be elucidated only in conjuction with concomitant effects on the fluorescence of a proximal polarity probe, such as the ANS chromophore. Despite the dramatic effects of the divalent metal cations on the fluorescence yields of tryptophyl and of bound ANS, the excitation energy exchange between these two chromophores is only slightly altered (Fig. 6). On the basis of the Rm6 law of resonance excitation transfer (34), one might expect very substantial changes in the transfer rate for even slight changes in the interchromophore separation. One possibility, although remote, is to consider
272
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AND PAPAGEORGIOU
cation-induced changes in both separation and mutual orientation of the interacting chromophores, of such fortuitous magnitudes, as to be mutually compensating. Disregarding the possibility of an orientation change, we may deduce from Fig. 6 gnd on the basis of an assumed R. = 23.3 A, that Mg*+ shortens the distance from ;he tryptophyl to bound ANS by about 0.5 A only. We may, therefore, conclude tentatively that metal divalent cations do not generate short-range structural changes in the neighborhood of the ANS-binding site. REFERENCES 1. PAPAGEORGIOU,G. (ln74) in Bioenergetics of Photosynthesis (Govindjee, ed.) Academic Press, New York. 2. BRODY, S. S., ZIEGELMAIR, C. A., SAMUELS, A., AND BRODY, M. (1966) Plant Physiol. 41,1709-1714. 3. HOMANN, P. (1969) Plant Physiol. 44, 932-936. 4. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. 5. SUN, A. S. K., AND SAUER, K. (1972) Biochim. Biophys. Acta 256, 409-427. 6. MOHANTY, P. K., BRAUN, B. Z., AND GOVINDJEE (1973) Biochim. Biophys. Acta 292, 459-476. 7. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. 8. RURAINSKI, H. J., RANDLES, J., AND HOCH, G. E. (1971) Fed. Eur. Biochem. Sot. Lett. 13, 98-100. 9. MARSHO, T. V., AND KOK, B. (1974) Biuchim. Biophys. Acta 333, 353-365. 10. GROSS, E. L., DILLEY, R. A., AND SAN PIETRO, A. (1969) Arch. Biochem. Biophys. 134, 450-462. 11. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biothem. Biophys. 146, 337-347. 12. DILLEY, R. A., AND SHAVIT, N. (1968) B&him. Biophys. Acta 162, 86-96. 13. PAPACEORGIOU,G. (1971) in Proc. 2nd Int. Congr. Photosynth. Res. (Forti, G., Avron, M., and Melandri, A. eds), Vol. 2, pp. 1535-1544, Dr. W. Jung, Publ., The Hague.
14. PAPACEORGIOU, G., AND ARGOUDELIS, C. (1973) Arch. Biochem. Biophys. 156, 134-142. 15. GROSS, E. L., AND HESS, S. C. (1974) Biochim. Biophys. Acta 339, 334-346. 16. FLEISCHER, S., FLEISCHER, B., AND STOECKENIUS, W. (1967) J. Cell Biol. 32, 193-208. 17. JI, T. H., HESS, T. L., AND BENSON, A. A. (1968) Biochim. Biophys. Acta 150, 676-685. 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 19. KLEIN, S. M., AND VERNON, L. P. (1974) Photothem. Photobiol. 19, 43-49. 20. DWSENS, L. N. M. (1952) PH.D. Thesis, University of Utrecht. 21. PARKER, C. A., AND REES, W. T. (1960) Analyst 85, 587-600. 22. WEBER, G., AND YOUNG, L. B. (1964) J. Biol. Chem. 239, 1415-1423. 23. BROCKLEHURST,J. R., FREEDMAN, R. B., HANCOCK, D. J., AND RADDA, G. K. (1970) Biochem. J. 116, 721-731. 24. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 25. HUANG, J.-S., HUANG, P.-Y., AND GOODMAN, R. N. (1973) Amer. J. Bot. 60, 80-85. 26. BRAND, L., AND WITHOLT, B. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 776-856, Academic Press, New York. 27. ANDERSON, R., AND WEBER, G. (1965) Biochemistry 4, 1949-1957. 28. WALLACH, D. F. H., FERBER, E., SELIN, D., WEIDEKAMM, E., AND FISCHER, H. (1970) Biochim. Biophys. Acta 203, 67-76. 29. TURNER, D. C., AND BRAND, L. (1968) Biochemistry 7, 3381-3390. 30. IZAWA, S., AND GOOD, N. E. (1966) Plant Physiol. 41,533-543. 31. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 32. WEBER, P. (1962) 2. Naturforsch. 17B, 683-688. 33. KRONMAN, M. J., AND HOLMES, L. G. (1971) Photochem. Photobiol. 14, 113-134. 34. F~RSTER, T. (1951) in Fluoreszenz Organischer Verbindungen, pp. 83-86, Vandenhoeck and Ruprecht, GSttingen.
