ARCHIVES
OF
BIOCHEMISTRY
interactions
AND
BIOPHYSICS
of Metal Cations
JOAN ISAAKIDOU’ Department
175,
of Biology,
541-548
(19761
with Lipid-Depleted
AND
Chloroplasts
GEORGE PAPAGEORGIOU
Nuclear Research Center “rDemocritos,”
Athens,
Greece
Received December 8, 1975 Lipid-depleted chloroplast membranes interact with metal cations in a manner that increases the fluorescence yield of the extrinsic chromophore 1-anilinonaphthalene-8sulfonate (ANS). The corresponding dissociation constants for the membrane complexes of Mg2+, Ca2+, and Mn2+ are 2 mM, but they can be increased to 7.2-7.4 mM in the presence of a second cation species. K+ and Na+ attach to lipid-free membranes with dissociation constants of 110 mM. The monovalent cations inhibit the binding of the divalent cations to the membrane noncompetitively. Since protein configuration around the ANS-binding site is not affected by metal cations [Isaakidou, J., and Papageorgiou, G. (1975) Arch. B&hem. Biophys. 168, 266-2721, structural changes on a larger scale, such as the contact of protein subunits, may underlie the cation-induced fluorescence rise. The divalent cations may facilitate such contacts by means of salt bridges, while the monovalent cations may facilitate them through surface charge neutralization. It is likely that the measured dissociation constants, in the case of divalent cations, correspond to the second step of the membrane-cation interaction, since only the completion of the salt bridge brings about the structural change reported by ANS. Monovalent cations weaken both bonds of a divalent with the membrane and, accordingly, their interference acquires a noncompetitive character. The results are discussed in relation to the role of proteins and of metal cations in enabling membranes to contact each other.
Several structural and functional characteristics of chloroplasts in vitro are related to the binding of metal cations to thylakoids. They include nonosmotic shrinkage (I, 2), stacking of thylakoids (35), lateral motion of lipoprotein particles (6-Q increased uptake of lipid soluble quenchers of chlorophyll fluorescence (9, 10) and spin probes (7)) reduced permeability to protons (ll), and stimulation of bound ANS2 fluorescence (5, 12). Cations are also required for phosphorylation-coupled electron transport (13-16), the Emerson enhancement effect (17), enhanced electron flow through P700 (18), and maximized photosystem II fluorescence (19-23). Certain phenomena, such as the coupling of electron transport to phosphoryla-
tion (13-16) and the Emerson effect (17), can be brought about either by low concentrations of divalent or by higher concentrations of monovalent cations. In other instances, and below a threshold concentration of monovalent cations (ca. 1 mM), a monovalent-divalent antagonism has been demonstrated. Notable examples of this are (a) the reversal by divalent cations of the inhibition of chloroplast fluorescence in the presence of low concentration of monovalent cations (24); and (b) the unstacking of thylakoids in the presence of a low concentration of monovalent cation salt and the restacking effect of divalent cations (25). Both lipids and proteins have been postulated as contributors to the fixed negative charges to which cations are supposed to bind (26). While lipids are implicated by inference only, the role of proteins is supported by the observation of Berg et al. (27) that conversion of the free carboxyls to N-
’ This work was carried out in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Miss J. Isaakidou. * Abbreviations used: ANS, l-anilinonapthalene8-sulfonate, NaDodSO1, sodium dodecyl sulfate. 541 Copyright All rights
0 1976 by Academic F’rees, Inc. of reproduction in any form reserved.
542
ISAAKIDOU
AND
substituted amides amounts to a nearly complete elimination of the membrane surface charge. In the present work, we seek further insight on the interaction of metal cations with membrane proteins. Our approach consists of monitoring the fluorescence of ANS-labeled chloroplasts that have been depleted of their lipids by means of the acetone-water-ammonia extraction procedure of Fleischer et al. (28). This extraction preserves membrane structures in mitochondria (28) and chloroplasts (29). Lipid-free chloroplasts bind ANS, whose fluorescence yield is stimulated by metal cations (30). MATERIALS
AND
PAPAGEORGIOU
0
!A,, 0
, I
-I
2 TIME
3
L
5
rmn
METHODS
Lipid-depleted membranes from spinach chloroplasm were prepared as described previously (30). These preparations are free of chlorophyll, and nearly free of carotenoids, and they consist essentially of structural protein with major polypeptides of molecular weight 23,000 and 60,000 (31). The membranes were washed twice with 2 mM tris(hydroxymethy1) aminomethane hydrochloride (Tris), pH 7.4 and 250 mM sucrose and were stored at -20°C until used. Samples were prepared by resuspending the membranes in the same medium, whose low Tris content minimized the interference of the amino group with the binding of the metal cations under study. ANS, Na-salt (Eastman-Kodak Co., Rochester, N.Y.) was recrystallized twice as the Mg-salt, and it was introduced to the samples to a final concentrawere carried out with the tion of 20 PM. Titrations chlorides of the studied cations and with delipidated chloroplast samples containing 0.1 mg protein/ml. Ten-minute incubation with the added salt, at room temperature, sufficed for stabilized ANS fluorescence signals. Fluorescence was excited at 360-390 nm, and it was observed at 480 nm, with a half-bandwidth of 46 nm. The observation band is on the long wavelength side of the emission maximum of membranebound ANS (458 nm; Ref. 30). Protein was measured according to Lowry et al. (32). All measurements were performed with samples that had been equilibrated at room temperature (23-25°C). RESULTS
Figure 1 depicts the kinetics traced by ANS fluorescence on adding the dye first and the salts subsequently to suspensions of lipid-free chloroplasts. Since ANS fluoresces very weakly in aqueous environment (4 = 0.004; Ref. 33) and quite
TIME
, min
FIG. 1. The kinetics of ANS fluorescence following the addition of the dye and of salts to buffered suspensions of lipid-free chloroplasts. Samples contain 0.1 mg protein/ml and 20 pM ANS. Salts are introduced to a final concentration of 50 mM in the case of monovalent cations, and 10 mM in the case of the divalent. Fluorescence excitation: 360-390 nm; detection, 480 nm, AA = 4-6 nm. For further spectroscopic details consult (30).
strongly when bound to the lipid-free membrane (4 = 0.121; Ref. 30), the observed signal should originate from the protein-bound population. A dramatic stimulation of this fluorescence is registered upon addition of 10 mM divalent metal chlorides to the suspension. Efficiencies vary with the cation species and are ranked in the order Mn2+ > Ca2+ > Mg2+ (Fig. 1, top). Monovalent cations stimulate the fluorescence of ANS to a lesser degree. Equal stimulations were obtained with 50 mM KC1 or NaCl (Fig. 1, bottom). Further addition of 50 mM monovalent metal chlorides, on top of the already present 10 mM divalent metal salt, induces a decrease of ANS fluorescence (Fig. 1, top). If, on the other hand, 10 mM divalent metal salt is added after the addition of 50 mM NaCl or KCl, a further increase of the
INTERACTION
OF CATIONS
-2
543
CHLOROPLASTS
ments, the latter enhancement was found to range between 0.35 and 0.45 of that induced by MgCl,. Figure 4 examines the antagonistic effect of monovalent cations on the stimulation of the fluorescence of membranebound ANS by divalent cations. MgCl, (10 mM) sufhces for nearly maximal stimulation (cf. Fig. 2). When NaCl is titrated into this sample, one observes a gradual de-
fluorescence signal is elicited (Fig. 1, bottom). In either case, the final intensities are about equal regardless of the order in which the cations have been added to the membrane suspension. When metal chlorides are titrated into ANS-labeled suspensions of delipidated chloroplasts, the fluorescence increases hyperbolically with the concentration of the added cation (Fig. 2). Threshold salt concentrations for maximal enhancement are 6-10 mM for divalent and 80-100 mM for monovalent cations. These results can be represented conveniently in terms of double reciprocal plots, such as those shown in Fig. 3. Dissociation constants calculated from the abscissa intercepts of this figure are KD = 2 mM for the divalent cations andI(, = 110 mM for the monovalent cations. Due to the shape of the concentration curves of Fig. 2, maximal fluorescence enhancement should correspond to the state of compelte occupation of the membrane sites by the added cation. This condition is certainly satisfied at infinite salt concentration. Accordingly, the maximal cationinduced enhancements of ANS fluorescence can be set proportional of the inverse ordinate intercepts of Fig. 3. Relative magnitudes of the maximal enhancements are 1, 1.17, and 1.63 for the salts MgCI,, CaCl,, and MnCl, (Fig. 3A). In contrast, Fig. 3B indicates the same maximal enhancement for both NaCl and KCl. In several experi-
yJ
WITH
2.5
1.0 0
2
4
6
8
IO
I2
0
20
40
60
80
100
120
UemM 140mM
[sALT]~',~
2
4
M'CI
FIG. 2. The cation-induced increase of ANS fluorescence as a function of the concentration of the added metal chloride. F, and F are the fluorescence intensities of membrane-bound ANS, in the absence and in the presence of the indicated electrolyte.
, ,” 0
JNI’Cl*
6
-
x IO
FIG. 3. Double reciprocal plots of the cation-induced against the concentration of the added salt. (A) divalent Other details, as in the legends to Figs. 1 and 2.
increments of ANS fluorescence cations; (B) monovalent cations.
544
ISAAKIDOU
AND
PAPAGEORGIOU
fluorescence emission in the manner described by the lower unmarked plot of the figure. When Mg2+ is present, the Mn2+induced enhancement is smaller and inversely related to the concentration of the interfering cation. The interference diminishes along with the rising concentration
MgCIz
I 0
20
40
60 N&l,
80
100
120
mM
4. The reversal of the MgCl,-induced increase of ANS fluorescence by NaCl. F, and F are the fluorescence intensities of membrane-bound ANS, in the absence and in the presence of NaCl. MgCl*, 10 mM, is always present in the membrane suspension. Other details, as in the legend to Fig. 1. FIG.
crease of the fluorescence signal, which levels off at about 100 mM concentration of the added salt. The NaCl-induced quenching amounts to 17% of the total fluorescence and to 33% of the Mg2+-induced increment. The effect is manifested only when divalent cations are present. However, their stimulation of ANS fluorescence is never quantitatively reversed by monovalent cations. Identical results were obtained with KCl. Figure 5 examines the interactions of divalent cations with the lipid-free membranes in the presence of sufficient monovalent metal salt to ensure maximal antagonistic effect. Under these conditions, the dissociation constants, which are read from the abscissa intercepts, have increased to 7.2 mM from the value of 2 mM obtained in the absence of monovalent cations (Fig. 3A). Maximal enhancement induced by divalent cations is also less, amounting to 25-40% of that observed in the absence of monovalent cation salts. These results imply noncompetitive interference, inasmuch as both the number of available membrane sites and their afEnity toward divalent cations are reduced in the presence of monovalent cations. Figure 6 indicates that mutual interference by divalent cations is expressed in a strikingly different way from that between monovalent and divalent cations. Titration of MnCl, into an ANS-labeled suspension of lipid-free chloroplasts stimulates
I
/
No K.
o
[SALT]-', m~-l x IO
FIG. 5. Double reciprocal plots of the ANS fluorescence increase with divalent cation chlorides, in presence of either 100 mM NaCl (open circles) or 100 mu KC1 (solid circles). Other details, as in the legends to Figs. 1 and 2.
[Mn C12];mM-1 FIG. 6. Double reciprocal plots of the ANS fluorescence increase with MnCl*, in the absence, as well as in the presence of 1 and 5 mM MgCl,. F,,,,,, ANS fluorescence at infinite MnCl,; F,,, ANS fluorescence in the absence of MnCl,; F, ANS fluorescence at a given, finite concentration of MnCl*.
INTERACTION
OF CATIONS
of Mn2+ and vanishes altogether at about 17 mM, the concentration that corresponds to the intersection point of the three plots in Fig. 6. The convergence of the three plots in Fig. 6 to the same point implies competitive interference, since it shows that the number of membrane sites available to Mn2+ does not change in the presence of Mg2+ ions. They do reduce, however, the affinity of the membrane to Mn2+, as suggested by the larger dissociation constants (i.e., shorter abscissa intercepts). From experiments similar to that of Fig. 6, we have calculated the dissociation constants of the membrane complexes with Mg2+, Ca2+, and Mn2+, when a second interfering cation is present. Inspection of Table I indicates an upper limit of 7.2-7.4 mM for the dissociation constants of all three divalent cations. This is obtained either with 100 mu NaCl (or KCl) or 10 mM of a second divalent metal chloride. Due to the large fluorescence increase at low concentrations of MnCl, (cf. Fig. 2), we have been unable to examine its interference in the binding of other divalent cations. Hence, this case is not included in Table I. TABLE
I
DISSKIATION CONSTANTS OF THE COMPLEXES OF DIVALENT METAL CATIONS WITH LIPID-FREE CHL~ROPLASTS IN THE PRESENCE AND IN THE ABSENCE OF OTHER CATIONS Additions (mM)
K,”
(mM)
Mg*+
CaZ+
Mn2+
2.0
2.0
2.0
2.4 3.0 4.1 1.2
2.4 3.0 4.1 7.2
2.4 3.0 4.1 7.2
1
-
2.5 5 10
-
3.6 4.7 6.5 7.4
3.6 4.7 6.5 7.4
4.2 4.8
-
4.2 4.8
None NaCl 10
20 50 100
M&l,
CaC12 0.5
2.0
u The K, values were calculated from the abscissa intercepts of plots such as those of Fig. 6. Other details as in the legend of Fig. 1.
WITH
CHLOROPLASTS
545
DISCUSSION
Before we proceed to interpret our results, we would like to qualify the physical system under study, i.e., the nature of the lipid-free membrane and the part of it reported by ANS fluorescence. On the basis of similar polypeptide patterns (as resolved by NaDodS04-acrylamide gel electrophoresis) the lipid-free residue of the acetone-water-ammonia extraction has been identified with the structural protein of chloroplast lamellae (30, 31). This extraction has been shown to preserve the inner membrane structures of mitochondria (28) and chloroplasts (29). Accordingly, we may visualize the lipidfree membrane as a two-dimensional structure of intrinsic proteins that are held together by lateral hydrophobic forces. The proteins expose the same surface, as in the native membrane, since delipidation does not seem to destroy their ability to stack (29). ANS binds to lipid-free chloroplasts to the extent of 126 nmol/mg protein, or 4 molecules per 32,000 molecular weight unit. As an anion, ANS should preferably line up at hydrophobic-hydrophilic interfaces of the membrane surface. Cations have been shown to stimulate the fluorescence of ANS by raising the quantum yield of the bound dye, although contributions from increased binding cannot be ruled out (30). Since ANS fluoresces more strongly in nonpolar media (33), we shall assume (in line with the accepted practice) that it reports on the hydrophobicity of its neighborhood in the membrane. The latter should be defined as a region not exceeding 40-60 A in diameter (34). It is known that ANS binds near a tryptophyl radical and that metal cations do not change the distance between the two chromophores (30). Hence, it appears that metal cations of the increase the hydrophobicity membrane surface without short-range structural effects around the ANS-binding site. The linearity of the plots in Fig. 3A indicates that ANS reports on one divalent cation binding site predominantly, for which the three tested cations compete (Fig. 6). One predominant site is indicated
546
ISAAKIDOU
AND
also for K+ and Na+ (Fig. 3B). Since the latter interfere with the divalent cations noncompetitively, we must assume either a distinct membrane site, or a distinct mode of action for the cations of each valence group. Relevant to this analysis is the question of whether the kinetically derived dissociation constants of divalent cations (Table I) correspond to the first, or to the second, interaction with singly ionized negative sites (such as side chain carboxy1 groups; 27) during the formation of a salt bridge. Divalent cations are thought to facilitate stacking of thylakoids by establishing salt bridges between subunits of adjacent membranes, while the monovalent enable membrane contact by shielding the negative charge and suppressing the coulombic repulsion (26). Since they do not change the structure of the ANS neighborhood, it follows that the divalent cations increase the quantum yield of the bound dye by means of a grosser effect, such as the interlinking of protein subunits of the same, or of adjacent membranes. In line with this reasoning, the kinetically determined dissociation constants should reflect the second step of the salt bridge formation, since it is this reaction that brings about the structural change which makes the ANSbinding sites less polar. Competition (Fig. 6) should also manifest at the second step interaction for both divalent cations since (i) it is reported by the ANS fluorescence, and (ii) it is different from the noncompetitive interference by monovalent cations. Monovalent cations probably shield both negative sites that can be linked by a divalent cation. Since one of these sites is not reported by the dye fluorescence (first step divalent cation-membrane interaction), while the other is reported (second step), interference by monovalent cations acquires a noncompetitive character. This occurs in spite of the fact that cations of both valence groups may attach to the same fixed anions, such as carboxyl groups. It is expected that at sufficiently high concentrations, both valence groups should suppress the membrane affinity for a tested divalent cation to the same extent, since they shield ultimately the same nega-
PAPAGEORGIOU
tive sites. This may account for our finding that the limiting dissociation constants for the membrane complexes of MI?+, Ca2+, and Mg2+, in the presence of interfering salts, are identical for both monovalent and divalent cations (i.e., 7.2-7.4 mM; cf. Table I). Several similarities, worthy of notice, exist between intact and delipidated chloroplasts relative to their responses to electrolytes. In both cases, electrolytes make the membrane surface more hydrophobic, as suggested by the formation of grana stacks (3-5) and by the stimulation of ANS fluorescence (5, 12, 30). Concentration thresholds for maximal cation effect range from 1 to 10 mM in the case of divalent metal salts and from 50 to 100 mM in the case of monovalent metal salts. This is true for the enhancement of ANS fluorescence in intact (5) and in lipid-free chloroplasts (Fig. 2), for the coupling of electron transport to phosphorylation (13), and for the maximization of the Emerson enhancement effect (17). Notice also should be taken of the fact that Mn2+ is the most potent divalent cation both in stimulating the chlorophyll fluorescence of intact chloroplasts (21) and the ANS fluorescence of lipid-free preparations (Figs. 2 and 3A). The analogy is probably fortuitous since divalent cations stimulate chlorophyll fluorescence by binding to the inside face of the thylakoid membrane (35,36), whereas ANS reports on the outer face. Perhaps, the ranking order of cations relative to these effects derives from a common mechanism of action, namely, the interlinking of protein subunits with salt bridges. On the basis of experiments with 45Ca2+, Gross and Hess (37) have identified two sites for divalent cations on the intact chloroplast membrane. The respective binding numbers and dissociation constants are 0.65 pmollmg chlorophyll and 8 + 3 PM for site I, and 0.5 f 0.2 pmol/mg chlorophyll and 51 ? 8 PM for site II. The second site was correlated with the divalent cationinduced structural changes that are supposed to generate increases in chlorophyll fluorescence and 540 nm turbidity. To decide whether the divalent catjon
INTERACTION
OF CATIONS
binding site of the lipid-free membrane may correspond to one of the above two sites, we have to consider the following. (a) The methodology employed in our work reports only on events that have an effect on the neighborhood of bound ANS. Binding of cations to sites that exert no such effect remains unreported. (b) The dissociation constants that have been measured with intact membranes are mixed values, since the radioactivity method cannot differentiate between the first and the second reaction occurring during the formation of a salt bridge. On the other hand, as was argued above, the fluorescence of ANS probably reports on the second reaction. (c) There is evidence that divalent cations induce increases in chlorophyll fluorescence only when they bind to the inner face of the thylakoid (35,36), suggesting that site II of Gross and Hess may be situated there. As an amphipathic molecule, however, ANS probably reports on the outer surface of the lipid-free membrane. In view of these considerations, therefore, it is not possible to correlate unambiguously the divalent cation binding site reported here with those previously reported by Gross and Hess (37). In conclusion, we have shown that divalent cations make the surface of chloroplast lamellae more hydrophobic, probably by the interlinking of protein subunits of the same, or of adjacent membranes. Monovalent cations also facilitate contacts, but in an entirely different and kinetically distinguishable manner. We would like to think that these results add to our understanding of the mechanism by which chloroplast lamellae are brought to contact. REFERENCES 1. GROSS, E. L., AND PACKER, L. (1967) Arch. Biothem. Biophys. 121, 779-789. 2. DILLEY, R. A., AND ROTHSTEIN, A. (1967) Biochim. Biophys. Acta 135, 427-443. 3. IZAWA, S., AND GOOD, N. E. (1966)PZantPhysiol. 41, 544-552. 4. ANDERSON, J. M., AND VERNON, L. P. (1967) Biochim. Biophys. Acta 143, 363-376. 5. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biochem. Biophys. 146, 337-347. 6. WANG, A.-I., AND PACKER, L. (1973) Biochim. Biophys. Acta 305, 488-492. 7. TORRES-PEREIRA, J., MELHORN, R., KEITH, A.
WITH
8.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30. 31.
CHLOROPLASTS
547
D., AND PACKER, L. (1974) Arch. Biochem. Biophys. 160, 90-99. OZAKIAN, G. K., AND SATIR, P. (1974) Proceed. D., AND PACKER, L. (1974) Arch. Biochem. Biophys. 160, 90-99. Nat. Acad. Sci. USA 71, 2052-2056. PAPAGEORGIOU, G. (1972) in Second International Congress on Photosynthetic Research, Stresa, Italy (Forti, G., Avron, M., and Melandri, A. B., eds.), pp. 1535-1544, Dr. W. Junk, N.V., The Hague. PAPAGEORGIOU, G., AND ARGOUDELIS, C. (1973) Arch. Biochem. Biophys. 156, 134-142. DILLEY, R. A., AND SHAVIT, N. (1968) B&him. Biophys. Acta 162, 86-96. VANDERMEULEN, D. L., AND GOVINDJEE (1974) Biochim. Biophys. Acta 368, 61-70. JAGENDORF, A. T., AND SMITH, M. (1962) Plant Physiol. 37, 135-141. AVRON, M., AND SHAVIT, N. (1963) Nat. Acad. Sci. -Nat. Res. Council Publ. 1145, p. 611. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. WALZ, D., SCHULDINER, S., AND AVRON, M. (1971) Eur. J. Biochem. 22, 439-444. SUN, A. S. K., AND SAWER, K. (1972) Biochim. Biophys. Acta 256, 409-427. RURAINSKI, H. J. AND HOCH, G. (1972) in Second International Congress on Photosynthetic Research, Stresa, Italy (Forti, G., Avron, M., and Melandri, A. B., eds.), pp. 133-141, Dr. W. Junk, N. V., The Hague. HOMANN, P. H. (1969) Plant Physiol. 44, 932936. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. MURATA, N., TASHIRO, H., AND TAKAMIYA, A. (1970) Biochim. Biophys. Acta 197, 250-256. WYDRZYNSKI, T., GROSS, E. L., AND GOVINDJEE (1975) B&him. Biophys. Acta 376, 151-161. BRIANTAIS, J.-M., VERNOTTE, C., AND MOYA, I. (1973) Biochim. Biophys. Acta 325,530-538. GROSS, E. L., AND HESS, S. C. (1973) Arch. Biothem. Biophys. 159, 832-836. GROSS, E. L., AND PRASHER, S. H. (1974) Arch. Biochem. Biophys. 164, 460-468. ANDERSON, J. M. (1975) B&him. Biophys. Acta 416, 191-235. BERG, S., DODGE, S., KROGMANN, D. W., AND DILLEY, R. A. (1974) Plant PhysioZ. 53, 619627. FLEISCHER, S., FLEISCHER, B., AND STOECKENIUS, W. (1967) J. Cell Biol. 32, 193-208. HUANG, J.-S., HUANG, P.-Y., AND GOODMAN, R. N. (1973) Amer. J. Bot. 60, 80-85. IBAAKIDOU, J., AND PAPAGEORGIOU, G. (1975) Arch. Biochem. Biophys. 168, 266-272. KLEIN, S. M., AND VERNON, L. P. (1974) Photothem. Photobiol. 19, 43-49.
548
ISAAKIDOU
AND PAPAGEORGIOU
32. LOWRY, 0. H., RQSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193,
265-275. 33. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 34. WEBER, G. (1970)in Spectroscopic Approaches to Biomolecular Conformation (Urry, D. W., ed.), pp. 23-31, American Medical Associa-
tion, Chicago. 35. KRAUSE, G. H. (1974) Biochim. Biophys. A&
333, 301-313. 36. MILLS, K., AND BARBER, J. (1975) Arch. Biothem. Biophys. 170, 306-314. 37. GROSS, E. L., AND HESS, S. C. (1974) Arch. Biohem.
Biophys.
339, 334-346.
OF
BIOCHEMISTRY
interactions
AND
BIOPHYSICS
of Metal Cations
JOAN ISAAKIDOU’ Department
175,
of Biology,
541-548
(19761
with Lipid-Depleted
AND
Chloroplasts
GEORGE PAPAGEORGIOU
Nuclear Research Center “rDemocritos,”
Athens,
Greece
Received December 8, 1975 Lipid-depleted chloroplast membranes interact with metal cations in a manner that increases the fluorescence yield of the extrinsic chromophore 1-anilinonaphthalene-8sulfonate (ANS). The corresponding dissociation constants for the membrane complexes of Mg2+, Ca2+, and Mn2+ are 2 mM, but they can be increased to 7.2-7.4 mM in the presence of a second cation species. K+ and Na+ attach to lipid-free membranes with dissociation constants of 110 mM. The monovalent cations inhibit the binding of the divalent cations to the membrane noncompetitively. Since protein configuration around the ANS-binding site is not affected by metal cations [Isaakidou, J., and Papageorgiou, G. (1975) Arch. B&hem. Biophys. 168, 266-2721, structural changes on a larger scale, such as the contact of protein subunits, may underlie the cation-induced fluorescence rise. The divalent cations may facilitate such contacts by means of salt bridges, while the monovalent cations may facilitate them through surface charge neutralization. It is likely that the measured dissociation constants, in the case of divalent cations, correspond to the second step of the membrane-cation interaction, since only the completion of the salt bridge brings about the structural change reported by ANS. Monovalent cations weaken both bonds of a divalent with the membrane and, accordingly, their interference acquires a noncompetitive character. The results are discussed in relation to the role of proteins and of metal cations in enabling membranes to contact each other.
Several structural and functional characteristics of chloroplasts in vitro are related to the binding of metal cations to thylakoids. They include nonosmotic shrinkage (I, 2), stacking of thylakoids (35), lateral motion of lipoprotein particles (6-Q increased uptake of lipid soluble quenchers of chlorophyll fluorescence (9, 10) and spin probes (7)) reduced permeability to protons (ll), and stimulation of bound ANS2 fluorescence (5, 12). Cations are also required for phosphorylation-coupled electron transport (13-16), the Emerson enhancement effect (17), enhanced electron flow through P700 (18), and maximized photosystem II fluorescence (19-23). Certain phenomena, such as the coupling of electron transport to phosphoryla-
tion (13-16) and the Emerson effect (17), can be brought about either by low concentrations of divalent or by higher concentrations of monovalent cations. In other instances, and below a threshold concentration of monovalent cations (ca. 1 mM), a monovalent-divalent antagonism has been demonstrated. Notable examples of this are (a) the reversal by divalent cations of the inhibition of chloroplast fluorescence in the presence of low concentration of monovalent cations (24); and (b) the unstacking of thylakoids in the presence of a low concentration of monovalent cation salt and the restacking effect of divalent cations (25). Both lipids and proteins have been postulated as contributors to the fixed negative charges to which cations are supposed to bind (26). While lipids are implicated by inference only, the role of proteins is supported by the observation of Berg et al. (27) that conversion of the free carboxyls to N-
’ This work was carried out in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Miss J. Isaakidou. * Abbreviations used: ANS, l-anilinonapthalene8-sulfonate, NaDodSO1, sodium dodecyl sulfate. 541 Copyright All rights
0 1976 by Academic F’rees, Inc. of reproduction in any form reserved.
542
ISAAKIDOU
AND
substituted amides amounts to a nearly complete elimination of the membrane surface charge. In the present work, we seek further insight on the interaction of metal cations with membrane proteins. Our approach consists of monitoring the fluorescence of ANS-labeled chloroplasts that have been depleted of their lipids by means of the acetone-water-ammonia extraction procedure of Fleischer et al. (28). This extraction preserves membrane structures in mitochondria (28) and chloroplasts (29). Lipid-free chloroplasts bind ANS, whose fluorescence yield is stimulated by metal cations (30). MATERIALS
AND
PAPAGEORGIOU
0
!A,, 0
, I
-I
2 TIME
3
L
5
rmn
METHODS
Lipid-depleted membranes from spinach chloroplasm were prepared as described previously (30). These preparations are free of chlorophyll, and nearly free of carotenoids, and they consist essentially of structural protein with major polypeptides of molecular weight 23,000 and 60,000 (31). The membranes were washed twice with 2 mM tris(hydroxymethy1) aminomethane hydrochloride (Tris), pH 7.4 and 250 mM sucrose and were stored at -20°C until used. Samples were prepared by resuspending the membranes in the same medium, whose low Tris content minimized the interference of the amino group with the binding of the metal cations under study. ANS, Na-salt (Eastman-Kodak Co., Rochester, N.Y.) was recrystallized twice as the Mg-salt, and it was introduced to the samples to a final concentrawere carried out with the tion of 20 PM. Titrations chlorides of the studied cations and with delipidated chloroplast samples containing 0.1 mg protein/ml. Ten-minute incubation with the added salt, at room temperature, sufficed for stabilized ANS fluorescence signals. Fluorescence was excited at 360-390 nm, and it was observed at 480 nm, with a half-bandwidth of 46 nm. The observation band is on the long wavelength side of the emission maximum of membranebound ANS (458 nm; Ref. 30). Protein was measured according to Lowry et al. (32). All measurements were performed with samples that had been equilibrated at room temperature (23-25°C). RESULTS
Figure 1 depicts the kinetics traced by ANS fluorescence on adding the dye first and the salts subsequently to suspensions of lipid-free chloroplasts. Since ANS fluoresces very weakly in aqueous environment (4 = 0.004; Ref. 33) and quite
TIME
, min
FIG. 1. The kinetics of ANS fluorescence following the addition of the dye and of salts to buffered suspensions of lipid-free chloroplasts. Samples contain 0.1 mg protein/ml and 20 pM ANS. Salts are introduced to a final concentration of 50 mM in the case of monovalent cations, and 10 mM in the case of the divalent. Fluorescence excitation: 360-390 nm; detection, 480 nm, AA = 4-6 nm. For further spectroscopic details consult (30).
strongly when bound to the lipid-free membrane (4 = 0.121; Ref. 30), the observed signal should originate from the protein-bound population. A dramatic stimulation of this fluorescence is registered upon addition of 10 mM divalent metal chlorides to the suspension. Efficiencies vary with the cation species and are ranked in the order Mn2+ > Ca2+ > Mg2+ (Fig. 1, top). Monovalent cations stimulate the fluorescence of ANS to a lesser degree. Equal stimulations were obtained with 50 mM KC1 or NaCl (Fig. 1, bottom). Further addition of 50 mM monovalent metal chlorides, on top of the already present 10 mM divalent metal salt, induces a decrease of ANS fluorescence (Fig. 1, top). If, on the other hand, 10 mM divalent metal salt is added after the addition of 50 mM NaCl or KCl, a further increase of the
INTERACTION
OF CATIONS
-2
543
CHLOROPLASTS
ments, the latter enhancement was found to range between 0.35 and 0.45 of that induced by MgCl,. Figure 4 examines the antagonistic effect of monovalent cations on the stimulation of the fluorescence of membranebound ANS by divalent cations. MgCl, (10 mM) sufhces for nearly maximal stimulation (cf. Fig. 2). When NaCl is titrated into this sample, one observes a gradual de-
fluorescence signal is elicited (Fig. 1, bottom). In either case, the final intensities are about equal regardless of the order in which the cations have been added to the membrane suspension. When metal chlorides are titrated into ANS-labeled suspensions of delipidated chloroplasts, the fluorescence increases hyperbolically with the concentration of the added cation (Fig. 2). Threshold salt concentrations for maximal enhancement are 6-10 mM for divalent and 80-100 mM for monovalent cations. These results can be represented conveniently in terms of double reciprocal plots, such as those shown in Fig. 3. Dissociation constants calculated from the abscissa intercepts of this figure are KD = 2 mM for the divalent cations andI(, = 110 mM for the monovalent cations. Due to the shape of the concentration curves of Fig. 2, maximal fluorescence enhancement should correspond to the state of compelte occupation of the membrane sites by the added cation. This condition is certainly satisfied at infinite salt concentration. Accordingly, the maximal cationinduced enhancements of ANS fluorescence can be set proportional of the inverse ordinate intercepts of Fig. 3. Relative magnitudes of the maximal enhancements are 1, 1.17, and 1.63 for the salts MgCI,, CaCl,, and MnCl, (Fig. 3A). In contrast, Fig. 3B indicates the same maximal enhancement for both NaCl and KCl. In several experi-
yJ
WITH
2.5
1.0 0
2
4
6
8
IO
I2
0
20
40
60
80
100
120
UemM 140mM
[sALT]~',~
2
4
M'CI
FIG. 2. The cation-induced increase of ANS fluorescence as a function of the concentration of the added metal chloride. F, and F are the fluorescence intensities of membrane-bound ANS, in the absence and in the presence of the indicated electrolyte.
, ,” 0
JNI’Cl*
6
-
x IO
FIG. 3. Double reciprocal plots of the cation-induced against the concentration of the added salt. (A) divalent Other details, as in the legends to Figs. 1 and 2.
increments of ANS fluorescence cations; (B) monovalent cations.
544
ISAAKIDOU
AND
PAPAGEORGIOU
fluorescence emission in the manner described by the lower unmarked plot of the figure. When Mg2+ is present, the Mn2+induced enhancement is smaller and inversely related to the concentration of the interfering cation. The interference diminishes along with the rising concentration
MgCIz
I 0
20
40
60 N&l,
80
100
120
mM
4. The reversal of the MgCl,-induced increase of ANS fluorescence by NaCl. F, and F are the fluorescence intensities of membrane-bound ANS, in the absence and in the presence of NaCl. MgCl*, 10 mM, is always present in the membrane suspension. Other details, as in the legend to Fig. 1. FIG.
crease of the fluorescence signal, which levels off at about 100 mM concentration of the added salt. The NaCl-induced quenching amounts to 17% of the total fluorescence and to 33% of the Mg2+-induced increment. The effect is manifested only when divalent cations are present. However, their stimulation of ANS fluorescence is never quantitatively reversed by monovalent cations. Identical results were obtained with KCl. Figure 5 examines the interactions of divalent cations with the lipid-free membranes in the presence of sufficient monovalent metal salt to ensure maximal antagonistic effect. Under these conditions, the dissociation constants, which are read from the abscissa intercepts, have increased to 7.2 mM from the value of 2 mM obtained in the absence of monovalent cations (Fig. 3A). Maximal enhancement induced by divalent cations is also less, amounting to 25-40% of that observed in the absence of monovalent cation salts. These results imply noncompetitive interference, inasmuch as both the number of available membrane sites and their afEnity toward divalent cations are reduced in the presence of monovalent cations. Figure 6 indicates that mutual interference by divalent cations is expressed in a strikingly different way from that between monovalent and divalent cations. Titration of MnCl, into an ANS-labeled suspension of lipid-free chloroplasts stimulates
I
/
No K.
o
[SALT]-', m~-l x IO
FIG. 5. Double reciprocal plots of the ANS fluorescence increase with divalent cation chlorides, in presence of either 100 mM NaCl (open circles) or 100 mu KC1 (solid circles). Other details, as in the legends to Figs. 1 and 2.
[Mn C12];mM-1 FIG. 6. Double reciprocal plots of the ANS fluorescence increase with MnCl*, in the absence, as well as in the presence of 1 and 5 mM MgCl,. F,,,,,, ANS fluorescence at infinite MnCl,; F,,, ANS fluorescence in the absence of MnCl,; F, ANS fluorescence at a given, finite concentration of MnCl*.
INTERACTION
OF CATIONS
of Mn2+ and vanishes altogether at about 17 mM, the concentration that corresponds to the intersection point of the three plots in Fig. 6. The convergence of the three plots in Fig. 6 to the same point implies competitive interference, since it shows that the number of membrane sites available to Mn2+ does not change in the presence of Mg2+ ions. They do reduce, however, the affinity of the membrane to Mn2+, as suggested by the larger dissociation constants (i.e., shorter abscissa intercepts). From experiments similar to that of Fig. 6, we have calculated the dissociation constants of the membrane complexes with Mg2+, Ca2+, and Mn2+, when a second interfering cation is present. Inspection of Table I indicates an upper limit of 7.2-7.4 mM for the dissociation constants of all three divalent cations. This is obtained either with 100 mu NaCl (or KCl) or 10 mM of a second divalent metal chloride. Due to the large fluorescence increase at low concentrations of MnCl, (cf. Fig. 2), we have been unable to examine its interference in the binding of other divalent cations. Hence, this case is not included in Table I. TABLE
I
DISSKIATION CONSTANTS OF THE COMPLEXES OF DIVALENT METAL CATIONS WITH LIPID-FREE CHL~ROPLASTS IN THE PRESENCE AND IN THE ABSENCE OF OTHER CATIONS Additions (mM)
K,”
(mM)
Mg*+
CaZ+
Mn2+
2.0
2.0
2.0
2.4 3.0 4.1 1.2
2.4 3.0 4.1 7.2
2.4 3.0 4.1 7.2
1
-
2.5 5 10
-
3.6 4.7 6.5 7.4
3.6 4.7 6.5 7.4
4.2 4.8
-
4.2 4.8
None NaCl 10
20 50 100
M&l,
CaC12 0.5
2.0
u The K, values were calculated from the abscissa intercepts of plots such as those of Fig. 6. Other details as in the legend of Fig. 1.
WITH
CHLOROPLASTS
545
DISCUSSION
Before we proceed to interpret our results, we would like to qualify the physical system under study, i.e., the nature of the lipid-free membrane and the part of it reported by ANS fluorescence. On the basis of similar polypeptide patterns (as resolved by NaDodS04-acrylamide gel electrophoresis) the lipid-free residue of the acetone-water-ammonia extraction has been identified with the structural protein of chloroplast lamellae (30, 31). This extraction has been shown to preserve the inner membrane structures of mitochondria (28) and chloroplasts (29). Accordingly, we may visualize the lipidfree membrane as a two-dimensional structure of intrinsic proteins that are held together by lateral hydrophobic forces. The proteins expose the same surface, as in the native membrane, since delipidation does not seem to destroy their ability to stack (29). ANS binds to lipid-free chloroplasts to the extent of 126 nmol/mg protein, or 4 molecules per 32,000 molecular weight unit. As an anion, ANS should preferably line up at hydrophobic-hydrophilic interfaces of the membrane surface. Cations have been shown to stimulate the fluorescence of ANS by raising the quantum yield of the bound dye, although contributions from increased binding cannot be ruled out (30). Since ANS fluoresces more strongly in nonpolar media (33), we shall assume (in line with the accepted practice) that it reports on the hydrophobicity of its neighborhood in the membrane. The latter should be defined as a region not exceeding 40-60 A in diameter (34). It is known that ANS binds near a tryptophyl radical and that metal cations do not change the distance between the two chromophores (30). Hence, it appears that metal cations of the increase the hydrophobicity membrane surface without short-range structural effects around the ANS-binding site. The linearity of the plots in Fig. 3A indicates that ANS reports on one divalent cation binding site predominantly, for which the three tested cations compete (Fig. 6). One predominant site is indicated
546
ISAAKIDOU
AND
also for K+ and Na+ (Fig. 3B). Since the latter interfere with the divalent cations noncompetitively, we must assume either a distinct membrane site, or a distinct mode of action for the cations of each valence group. Relevant to this analysis is the question of whether the kinetically derived dissociation constants of divalent cations (Table I) correspond to the first, or to the second, interaction with singly ionized negative sites (such as side chain carboxy1 groups; 27) during the formation of a salt bridge. Divalent cations are thought to facilitate stacking of thylakoids by establishing salt bridges between subunits of adjacent membranes, while the monovalent enable membrane contact by shielding the negative charge and suppressing the coulombic repulsion (26). Since they do not change the structure of the ANS neighborhood, it follows that the divalent cations increase the quantum yield of the bound dye by means of a grosser effect, such as the interlinking of protein subunits of the same, or of adjacent membranes. In line with this reasoning, the kinetically determined dissociation constants should reflect the second step of the salt bridge formation, since it is this reaction that brings about the structural change which makes the ANSbinding sites less polar. Competition (Fig. 6) should also manifest at the second step interaction for both divalent cations since (i) it is reported by the ANS fluorescence, and (ii) it is different from the noncompetitive interference by monovalent cations. Monovalent cations probably shield both negative sites that can be linked by a divalent cation. Since one of these sites is not reported by the dye fluorescence (first step divalent cation-membrane interaction), while the other is reported (second step), interference by monovalent cations acquires a noncompetitive character. This occurs in spite of the fact that cations of both valence groups may attach to the same fixed anions, such as carboxyl groups. It is expected that at sufficiently high concentrations, both valence groups should suppress the membrane affinity for a tested divalent cation to the same extent, since they shield ultimately the same nega-
PAPAGEORGIOU
tive sites. This may account for our finding that the limiting dissociation constants for the membrane complexes of MI?+, Ca2+, and Mg2+, in the presence of interfering salts, are identical for both monovalent and divalent cations (i.e., 7.2-7.4 mM; cf. Table I). Several similarities, worthy of notice, exist between intact and delipidated chloroplasts relative to their responses to electrolytes. In both cases, electrolytes make the membrane surface more hydrophobic, as suggested by the formation of grana stacks (3-5) and by the stimulation of ANS fluorescence (5, 12, 30). Concentration thresholds for maximal cation effect range from 1 to 10 mM in the case of divalent metal salts and from 50 to 100 mM in the case of monovalent metal salts. This is true for the enhancement of ANS fluorescence in intact (5) and in lipid-free chloroplasts (Fig. 2), for the coupling of electron transport to phosphorylation (13), and for the maximization of the Emerson enhancement effect (17). Notice also should be taken of the fact that Mn2+ is the most potent divalent cation both in stimulating the chlorophyll fluorescence of intact chloroplasts (21) and the ANS fluorescence of lipid-free preparations (Figs. 2 and 3A). The analogy is probably fortuitous since divalent cations stimulate chlorophyll fluorescence by binding to the inside face of the thylakoid membrane (35,36), whereas ANS reports on the outer face. Perhaps, the ranking order of cations relative to these effects derives from a common mechanism of action, namely, the interlinking of protein subunits with salt bridges. On the basis of experiments with 45Ca2+, Gross and Hess (37) have identified two sites for divalent cations on the intact chloroplast membrane. The respective binding numbers and dissociation constants are 0.65 pmollmg chlorophyll and 8 + 3 PM for site I, and 0.5 f 0.2 pmol/mg chlorophyll and 51 ? 8 PM for site II. The second site was correlated with the divalent cationinduced structural changes that are supposed to generate increases in chlorophyll fluorescence and 540 nm turbidity. To decide whether the divalent catjon
INTERACTION
OF CATIONS
binding site of the lipid-free membrane may correspond to one of the above two sites, we have to consider the following. (a) The methodology employed in our work reports only on events that have an effect on the neighborhood of bound ANS. Binding of cations to sites that exert no such effect remains unreported. (b) The dissociation constants that have been measured with intact membranes are mixed values, since the radioactivity method cannot differentiate between the first and the second reaction occurring during the formation of a salt bridge. On the other hand, as was argued above, the fluorescence of ANS probably reports on the second reaction. (c) There is evidence that divalent cations induce increases in chlorophyll fluorescence only when they bind to the inner face of the thylakoid (35,36), suggesting that site II of Gross and Hess may be situated there. As an amphipathic molecule, however, ANS probably reports on the outer surface of the lipid-free membrane. In view of these considerations, therefore, it is not possible to correlate unambiguously the divalent cation binding site reported here with those previously reported by Gross and Hess (37). In conclusion, we have shown that divalent cations make the surface of chloroplast lamellae more hydrophobic, probably by the interlinking of protein subunits of the same, or of adjacent membranes. Monovalent cations also facilitate contacts, but in an entirely different and kinetically distinguishable manner. We would like to think that these results add to our understanding of the mechanism by which chloroplast lamellae are brought to contact. REFERENCES 1. GROSS, E. L., AND PACKER, L. (1967) Arch. Biothem. Biophys. 121, 779-789. 2. DILLEY, R. A., AND ROTHSTEIN, A. (1967) Biochim. Biophys. Acta 135, 427-443. 3. IZAWA, S., AND GOOD, N. E. (1966)PZantPhysiol. 41, 544-552. 4. ANDERSON, J. M., AND VERNON, L. P. (1967) Biochim. Biophys. Acta 143, 363-376. 5. MURAKAMI, S., AND PACKER, L. (1971) Arch. Biochem. Biophys. 146, 337-347. 6. WANG, A.-I., AND PACKER, L. (1973) Biochim. Biophys. Acta 305, 488-492. 7. TORRES-PEREIRA, J., MELHORN, R., KEITH, A.
WITH
8.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30. 31.
CHLOROPLASTS
547
D., AND PACKER, L. (1974) Arch. Biochem. Biophys. 160, 90-99. OZAKIAN, G. K., AND SATIR, P. (1974) Proceed. D., AND PACKER, L. (1974) Arch. Biochem. Biophys. 160, 90-99. Nat. Acad. Sci. USA 71, 2052-2056. PAPAGEORGIOU, G. (1972) in Second International Congress on Photosynthetic Research, Stresa, Italy (Forti, G., Avron, M., and Melandri, A. B., eds.), pp. 1535-1544, Dr. W. Junk, N.V., The Hague. PAPAGEORGIOU, G., AND ARGOUDELIS, C. (1973) Arch. Biochem. Biophys. 156, 134-142. DILLEY, R. A., AND SHAVIT, N. (1968) B&him. Biophys. Acta 162, 86-96. VANDERMEULEN, D. L., AND GOVINDJEE (1974) Biochim. Biophys. Acta 368, 61-70. JAGENDORF, A. T., AND SMITH, M. (1962) Plant Physiol. 37, 135-141. AVRON, M., AND SHAVIT, N. (1963) Nat. Acad. Sci. -Nat. Res. Council Publ. 1145, p. 611. SHAVIT, N., AND AVRON, M. (1967) Biochim. Biophys. Acta 131, 516-525. WALZ, D., SCHULDINER, S., AND AVRON, M. (1971) Eur. J. Biochem. 22, 439-444. SUN, A. S. K., AND SAWER, K. (1972) Biochim. Biophys. Acta 256, 409-427. RURAINSKI, H. J. AND HOCH, G. (1972) in Second International Congress on Photosynthetic Research, Stresa, Italy (Forti, G., Avron, M., and Melandri, A. B., eds.), pp. 133-141, Dr. W. Junk, N. V., The Hague. HOMANN, P. H. (1969) Plant Physiol. 44, 932936. MURATA, N. (1969) Biochim. Biophys. Acta 189, 171-181. MURATA, N., TASHIRO, H., AND TAKAMIYA, A. (1970) Biochim. Biophys. Acta 197, 250-256. WYDRZYNSKI, T., GROSS, E. L., AND GOVINDJEE (1975) B&him. Biophys. Acta 376, 151-161. BRIANTAIS, J.-M., VERNOTTE, C., AND MOYA, I. (1973) Biochim. Biophys. Acta 325,530-538. GROSS, E. L., AND HESS, S. C. (1973) Arch. Biothem. Biophys. 159, 832-836. GROSS, E. L., AND PRASHER, S. H. (1974) Arch. Biochem. Biophys. 164, 460-468. ANDERSON, J. M. (1975) B&him. Biophys. Acta 416, 191-235. BERG, S., DODGE, S., KROGMANN, D. W., AND DILLEY, R. A. (1974) Plant PhysioZ. 53, 619627. FLEISCHER, S., FLEISCHER, B., AND STOECKENIUS, W. (1967) J. Cell Biol. 32, 193-208. HUANG, J.-S., HUANG, P.-Y., AND GOODMAN, R. N. (1973) Amer. J. Bot. 60, 80-85. IBAAKIDOU, J., AND PAPAGEORGIOU, G. (1975) Arch. Biochem. Biophys. 168, 266-272. KLEIN, S. M., AND VERNON, L. P. (1974) Photothem. Photobiol. 19, 43-49.
548
ISAAKIDOU
AND PAPAGEORGIOU
32. LOWRY, 0. H., RQSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193,
265-275. 33. STRYER, L. (1965) J. Mol. Biol. 13, 482-495. 34. WEBER, G. (1970)in Spectroscopic Approaches to Biomolecular Conformation (Urry, D. W., ed.), pp. 23-31, American Medical Associa-
tion, Chicago. 35. KRAUSE, G. H. (1974) Biochim. Biophys. A&
333, 301-313. 36. MILLS, K., AND BARBER, J. (1975) Arch. Biothem. Biophys. 170, 306-314. 37. GROSS, E. L., AND HESS, S. C. (1974) Arch. Biohem.
Biophys.
339, 334-346.
